Transcriptome-Wide Analysis of HuR Function Identifies TXNIP-Mediated Redox Dysregulation in Osteocytes

Post-transcriptional gene r egulation modulates gene expression by modulating RNA pr ocessing through RNA splicing, modifications, localization, stability , tr anslation and fat, 1,2,3,4,5,6 generating tr anscript variants that contribute to proteomic diver sity . Thus, post-transcriptional regulation plays a k ey role in str ess adaptation optimizing pr otein s ynthesis to pr omot e cell survival. 7,8 RNA binding proteins (RBPs) regulat e RNAs by binding structured RNA motif s to control nearly every aspect of RNA metabolism and function. 9,10,11,12 RBPs contribut e to tissue-specific disease phenotypes, often reflecting the expr ession pattern of their RNA t argets and cofactor s as well as the RBP's own abundance and act ivity level. 13 Alterations in RBPs or their targets ar e associated with diverse pathologies, including neurodegenerative, muscular , autoimmune, and metabolic diseases driven by impaired RNA transport, splicing, or clear ance, highlighting the critical role of RNA-RBPs int eractome in determining disease outcomes. 9,13,14 HuR [‘human antigen R’ , also known as ELAVL1 (embryonic lethal abnormal vision-lik e 1)] is a ubiquitously expr essed RNA-binding protein, a classic RBP , possessing RNA r ecognition motif s (RRM) and a k ey modulator of post transcriptional gene r egulation. 15 It has been extensively studied as a cancer drug t arget 16 and its global deletion has been shown t o be embryonically lethal, 17 and conditional knock outs experienced impaired post-natal formation of sk eletal, immune, and other t issue s. 18,19,20,21 Beyond development, tissue-specific knock downs disrupt metabolic and immune homeostasis; wher e glucose and lipid metabolism linking it to obesity and insulin resist ance. 22,23,24 In bone, an ovariectomy-induced bone loss model, had reduced HuR expr ession, wher eas overexpression alleviat ed bone loss and silencing impaired osteoblast diff erentiation. 25 In contr ast, diabetic mouse models showed increased HuR mRNA levels, and its knock down impr oved glucose metabolism and bone micr oarchitecture. 26 Similarly , in MC3T3-E1 cells exposed t o high glucose, HuR expression incr eased, and its silencing reduced apoptosis while promoting osteogenic diff erentiation. 26 T h e s e s t u d i e s d e m o n s t r a t e t h a t H u R e x p r e s s i o n a n d i t s e f f e c t s a r e t i s s u e a n d c o n t e x t dependent. Osteocyt es, comprising the majority of bone cells, ar e post mitotic mechanosensor s maint aining bone remodeling through orchestrating resorption and f ormation. They ar e endocrine and signaling cells that maintain sk eletal homeost asis. 27,28,29,30 T o date, post-transcriptional RNA regulation in osteocytes and the RBP s regulating it remain largely uncharted. Here, we perf ormed an unbiased RBP motif enrichment analysis on .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 22, 2025. ; https://doi.org/10.1101/2025.08.08.668956doi: bioRxiv preprint d i f f e r e n t i a l l y s p l i c e d e v e n t s f r o m R N A - s e q d a t a o f p r i m a r y o s t e o c y t e s f r o m D m p 1 - t opaz mice cultur ed under high-glucose conditions. W e introduce f or the first time the alternative splicing programs r egulat ed in osteocytes and map the RBPs that regulate them. We further reveal HuR as a k ey regulator during high glucose (HG) str ess. T o define its functional role, we employed an HuR knock down model and investigated both diff er ential gene expression and alt ernative splicing programs, ther eby homing in on HuR’ s role in sustaining cellular homeostasis, coor dinating translation, and supporting mitochondrial function. Our data further demonstrates that HuR acts on thioredoxin-inter acting protein (TXNIP) expression, a glucose-responsive inhibitor of the antio xidant thioredoxin and its silencing prot ect against oxidative str ess injur y . 31 Specifically , HuR directly binds TXNIP (mRNA) and regulates its st ab ility , thereby pr eserving redox balance. Collectively , these findings pr ovide mechanistic insight into HuR’ s function and highlight its potential role in pr eserving skelet al homeostasis in metabolic diseases.

Splicing-driven RBP motif analysis identifies HuR as a t op regulator in osteocytes under high glucose stress Primary osteocytes obtained fr om Dmp1-T opaz mice wer e cultured in HG or NG f or 72 hours. RNA-seq analy sis showed minimal gene expr ession changes with TXNIP and CD36 among the t op r egulat ed (Figure 1A). rMA T s analysis of the 5 canonical splicing pat terns; skipped ex on (SE), mutually ex clusive exons (MXE), retained intr ons (RI), alternative 5’ splice site (A5SS), and alt ernative 3’ splice site (A3SS) revealed significant changes (FDR 0.2) in ~2% of total events, predominantly SE and A3SS (Figur e 1B). 32 GO Biological Processes (GOBP) and pathway enrichment analysis of AS showed that diff erent AS programs c ontribute diff erently to cellular biology . Specifically , SE DSGs revealed enrichment in cyt osk eletal remodeling and protein regulation via ubiquitination, and several pathways involved in matrix maintenance and repair (Figure 1C, Supplementary T able 1). MXE DSGs were enriched in pathways involved in collagen s ynthesis and organization, skelet al tissue development and pathways involved in cell gr owth and r egulation of fluid balance (Figure 1D , Supplementary T able 1). A5SS DSGs were predominat ed by pathways r elated to post transcriptional and translational r egulation including ribosome t argeting, rRNA processing and quality con trol of mRNA translation. A3SS DSGs wer e involved in RNA splicing, sever al signaling pathwa ys involved in cell growth and diff erentiation and parathyroid hormone action. DSGs in RI showed cell cycle r elat ed terms, ribosome and translation r egulation as well as ubiquitination (Supplementary Fig 1A-C and Supplementary T able 1). .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 22, 2025. ; https://doi.org/10.1101/2025.08.08.668956doi: bioRxiv preprint B e c ause these AS chang es su g g es t ed alt er ed pos t-tr ans cript ional con t r ol, w e ne xt in t e rr og a t ed RBP moti f enrichment using r MAPS2. 33 W e filt er ed t he t o p up enriched and down enr i ched moti f p os it i o ns fr om a ll 5 AS dat asets ba sed on p values < 0.05 (t t est) . Ar ou nd upr egulat e d s plice ev en ts, HNR NPL and HuR w er e the t o p e nriched in SE ( F igur e 2A, Supplement ar y T a b l e 2), in MXE, ZC 3H14 a n d Hu R. In A5SS, HNRN PL and H uR, in A3SS, FU S and H N R N PH2 and in R I we obser v ed no e nr ic hed R BP m otif s a t a cut of f v alue of p <0 .05 (Supp l eme n t a r y Fig 2A -D and S up pleme n t a ry T able 2). Ar ound do w n r egula t ed spli ce ev e n ts, KHD R BS1 and Z C3H 14 w er e the t op enr iched in SE ( F igur e 2B ), in MXE, Z C 3H 14 and T r a2-bet a, in A5SS, HNR PLL and HuR show ed the highes t enrichmen t. In A 3SS , RBM47 and SRp20 w er e the mos t enr ic h ed while in RI R BMS3 and P ABPN1 w er e dominan t (Supplemen t a r y Fig 3A-D and Supplem en t a r y T able 2) . The analy sis s h owed t ha t sever al H uR mot i f s w er e enr ic hed in up r eg ula t ed s p l ice ev en ts. Fi gur e 2C show s an e xampl e of HuR b inding moti f s ar ound SE d i f f er e n tia lly s p l i c e d exo ns . O u r bi oi nfo r m at i c s a na l y s is p o i nt s to Hu R a s a to p r e g u l a t o r y R BP i n A S , t her e for e w e meas u r ed H uR mRNA a n d pr ot ein level s in ML O-Y4 cells e x p o s ed t o HG. mRN A e xpr ess ion wa s do wnr egulat e d as mea sur ed b y qP CR w hile pr ot ei n e x pr es sion did not c hang e as measur ed by w est ern b l o t (Figur e 3A-C) . Simila rly , HuR mRNA a lso decr eased in the long bones of h yp er gly cemic mi ce (Supplemen t a ry Fig 4) tha t w e r e f ed a moder a t ely HFD f or 16 w eeks as mea s u r ed by qP C R while pr ot ein l ev e ls did not chang e c ompar ed t o ND ( Fi gur e 3D- F). HuR KD r ev eal s tr anscript o mic an d phenot y pic chang es in ML O - Y4 c ells W e knoc k ed do w n HuR in MLO-Y4 ce lls (Y KD ) us ing t wo indep endent shRN A c ons truct s , bo th of whic h eff ectiv ely r educed HuR pr ot e in levels (Figur e 4A ). D es pit e co mpar able knoc k down e f ficiency , w e ob ser v ed d iff er ences bet w e en the tw o c ons tr uc t s . C onst ru ct A ( Y A KD ) ind uc ed s ev er e c yt ot o xic it y , including a signific an t los s of cell viabil it y (Figur e 4B ) a n d dr amatic morpho l o g ic al c han ge s c h a r ac t erized b y a s ev er ely r educed cyt oplasmic v olume and near co mpl et e loss of dendr it es (Fi gu r e 4C, D). Additionall y , G S E A r e s u l t s f r o m Y A KD s how ed enrichmen t i n DNA d amage and cell cy cle chec kpoin t pa t hw ay s in addition t o RNA met abolis m and s p l icing t er m enrichmen t espit e min imal evidence of diff er en t ial s p l icing (Supp lement ary Fig u r e A, 5B) . T h i s pa t t ern is con sist en t with g lob a l s tr es s r esponse r a ther than spec ific H uR-depende n t r egul a tion. These ph enotypic change s su g g est pot en tial of f -t ar g e t ef f ects. Co n str uct B (YB KD ) e xperienced r educ ed c ell viabi lity an d morp ho logical chang es t hough t o a less er e x t ent th a n Y A KD , and pr e served cell dend r i tes (Fi gu r e 4C, D ). T o ens ur e tha t ou r r esults w er e no t c onf ounded by pot e n tial o f f- t ar g et ef f ects in c onst ruct A , w e us ed Y B KD f o r f u r t h e r analy sis, her eaft er , t he t er ms HuR KD and YB KD ar e used in t er chang ea bly . R NA-seq .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 22, 2025. ; https://doi.org/10.1101/2025.08.08.668956doi: bioRxiv preprint analy sis of YB KD r evealed 129 dif f er en t ially e xpr ess ed g enes a t |Log2 F C| ≥ 1 and a d j u s t e d p v a l u e < 0 . 0 5 c o mp a r e d t o Mo c k KD i n c luding H uR downr eg u l a t ion (Figur e 4E). The t op 10 en r i ched pa thw a y s in pr e-r ank ed g ene se t en richment anal y s i s show ed do w n r egula tion o f e xtr acellula r mat r ix or g aniz a tion and r e model ing , inc lu ding s ev er al c ollag ens , ma t rix met al lopr o t einas es and ca t heps in s- gr owt h a n d d evelopme nt pa t hway s, f a t ty a cid and cholest er ol bios yn the si s pa thwa y s , in ad dition t o cell si gnalin g pa t hw ay s in v olved i n inflamma ti on and cellular hom eos t asis . Upr egula t ed pa thw a y s show ed a clear tr end t ow ar ds enric hmen t in pa thw a y s r ela t ed t o qualit y con t r ol o f mRN A pr oces s in g , t r an s la t i o n and rib o s omal t erms. In addit ion t o p at hw ay s r ela t ed to immunity and pa th wa y s r ela t ed t o c yt os k elet on o r g aniza tion (Fig u r e 4F and Supp l emen t a ry T able 3) . Ov er all, the tr anscript omic p r ofile of Hu R KD poin ts t o a c omple x r ol e f or HuR i n c ontr oll i n g sever al as pe cts of cellular ho meos t a s i s including cyt os k el et al or g aniz at ion, e xtr a cellul ar matrix p r oteins and a r ole in r egula ting m RNA pr ocessing and tr an sla tion. HuR r eg u la t es t r ansla tional and m et abolic st r ess adap t a tion splicing pr ogr am s W e fu rther c ompar ed alt ernat i ve m RNA s pli cing pa t t erns bet ween YB KD a n d M o c k KD . W e summar ized the output of t he A S analy s i s fr om junction a n d e x on body c oun t s ( JCE C), the t o t al numb er o f JC E C even ts was 24 th ous and ev en ts, with 3% s ho wing signific a n c e between YB KD and Moc k KD . A m on g th e A S p at te r ns d e te c te d , S E r epr esen t e d the mos t fr eq uen t ly r egula t ed ev e n ts, while RI even t s w er e the leas t r egulat ed ( F igur e 5A ). This o bs erva t ion aligns with the earl ier H uR moti f analysis in pr imary ost eoc yt es (Supplemen t ar y Fig 2 and 3) , wher e RI ev en ts show ed t he leas t enr ic h men t in HuR mo tif s ar ou nd diff er e n tia lly s pliced in t r ons. DS G s (Figur e 5B , Supp l emen t a ry T able 4) wer e us ed t o per f o rm g ene o n t o logy (G O) and over r epr esen ta tion fu nc t ional annota ti o n ( O R A) f o llowing HuR KD . The t o p 10 sc oring cellular c ompon en ts i n c luded ribonucleopr o t ein c omple xes (RN P s), rib osomes and mi t ochondria (Figur e 5C). ORA analy sis r esult s r ev ealed tw o major cl u st er s of enriched pa t hw ay s. Fi r st , t r an slat ion-relat ed t erms, incl u ding r RNA pr oces s ing, RNA spli cin g , mRN A sur v eilla n c e (nonsen s e- mediat ed d eca y), ribosomal subunit s, and tr ansla tional r egulat ion c ons is t en t with H uR’ s r ole as a n RBP . Se con d, met abolic or st ress-r es pon s e t erms, inc lud i n g aut ophagy , H IF-1α signal ing , and insulin si gnaling pa th wa y s (Figur e 5D , Supp l emen t a ry T able 4) . HuR KD sen s it i z es cells t o h yp er g ly cemic and o xida tiv e s tr ess induc ed c ell dea t h Our i n i t ial analy si s poin t ed t o Hu R motif as a t op enr ic hed motif ar ou nd s pliced t r ans c r ipts in HG c onditions, there f or e w e as sed the ef f ec t of H G on YB KD c e l l s . W e cultu r ed cells with N G or HG and M as o smot i c pr es s ur e c on tr o l , w e us ed H 2 O 2 a s a po s itiv e c on tr ol . H G ind uc ed cell dea th only in YB KD cells but not in Moc k KD aft er 48 and .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 22, 2025. ; https://doi.org/10.1101/2025.08.08.668956doi: bioRxiv preprint 72 hou r s indic a ting tha t H uR KD sens it ize s cells t o h yper gly cemic i n jur y . H y dr og en per o xi d e induced o xida tiv e cell death in b oth cel l lines; b ut wi t h mar k edly gr eat er let hal it y in YB KD comp a r ed to Mock KD ( F igur e 6A ). H G is kn own t o induce o xida tive s tr ess, ther e f or e, w e n ext e xamined whether these chang e s af f e c t ed r edo x ho meost as is and cell u l ar su sceptibilit y t o o xida tive str es s . W e perf or med D CFD A a s s a y which measur es R O S lev el s, t he r esults indic a t ed a decr ease in R OS pr od uction un der baseline c ult ur e co nditions in YB KD , howev er , upon tr ea ti n g cells with TBH P , a R O S g ener at ing c ompound, R O S a c c u mula tion dou bled in YB KD c ompar ed t o M oc k KD (Figur e 6B ). HuR dep le tion disrup ts o xida t iv e str es s a dapt a t ion by st abiliz in g TX N I P m RN A T o f urth er e xpl or e the mechanis m by whi ch H uR r e gu l a t es o x ida ti v e str e ss r es pon se, w e m e a s u r e d t h e e x p r e s s i o n l e v e l s o f T X N I P . T X N I P m R N A l e v e l s i n c r e a s e d a f t e r H u R knoc k down (Figur e 6C). We then perf ormed RNA i mmunop recipit a t ion (RIP) , which show ed t ha t H uR-speci f ic an tibody ef f ec t i v ely c o-pr ec ipit a t ed TXNIP mR NA compared t o neg a tiv e IgG c on tr ol and pr ecipit at ed 70% mo r e TX NI P mR N A th a n Mo ck KD c onfir ming dir ect int er action (Figur e 6D). We th en quan tified TX N I P mR NA deca y aft er actinom y cin D tr ea t ment; Hu R K D inc r eas ed TX NI P s t ability ( Δ t ½ = 1 . 4 7 0 → 1.846 ho ur s), an almost 25% inc r ea s e in mRNA half-li f e. T og eth er , H uR bind s TX N I P mR NA and limi ts its s t ability , pr ovid ing a mec hanis tic link bet ween HuR los s an d heig h t en ed o xida tive st r ess sen s it iv it y . HuR r eg u la t es in s ulin se nsitivity Our DS G analy si s poin t ed t o ins u l in s ignaling t er m enr ic hment, and in s ulin is a known suppr essor of T X NIP e xpr es si o n. 34 Ther e f o r e, we in v est ig at ed the functionalit y of o ur D SG analy s is (Figur e 5D ). W e as se s se d whether HuR KD alt er ed ins ulin r es po ns iv eness. H uR KD exhibit ed a r educed r esponse t o ins ulin s timula tion , a s evidenced b y d ecr ea sed A KT phosphoryla tion (p-AKT) in Figur e 6F . HuR KD induc es m it ochondr ial dysf u nc t ion Our D SG analy sis r ev ealed enrichmen t of mi t ochon drial -r el a ted t erms, and H uR KD cells s ho wed both heig h t ened suscept i b i li t y t o R O S-induced dea th and i nc r eas ed R OS ac cumula tion. Bec a u s e mit ochondr i a l r espir a tion is a pr i mary sour ce of cellular R O S, t hes e findings pr omp t ed us t o e xami n e mi t ochondr ial mas s and function. T o quan ti fy mi t ochondria l density , we u sed Mit ot r ac k er CM XR o s R ed, a sele ctiv e mi t ochondr i al dy e. Mit ochondr i a loc alized a r ound t he nuclei a n d e x t ended t o d endri t es, and w e obser v ed r educed mean fluo r es cence in t ensity in YB KD c e l l s c o m p a r e d t o M o c k KD ( Fi gu r e 7 A , B ) . Ne xt, w e measur ed m it oc h ondria l f unction using Seahor s e XF Cell Mito-St r ess t es t ( F igur e 7C). YB KD cells e xhibi t ed r educ ed mi t ochondr i al r es pir at ion. Basa l OCR, ma ximal .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 22, 2025. ; https://doi.org/10.1101/2025.08.08.668956doi: bioRxiv preprint r es pir at ion, s par e r e spir a t ory capaci ty , c oupling e f ficienc y and A T P p r od uction w er e r educed. Pr ot on leak r e ma ined unc han ged, su g g e st i n g t ha t th e m it oc h ondria l membr ane is in t ac t . Th e o bs erved r eduction in O C R al so e xt end ed t o n on - mi t ochondria l r es pir a tion, sug g es ting t ha t th e decl ine in o xyg en c onsump t i o n is partly a t t r ibut abl e t o reduced activity of non- mi t ochondr ia l o xy g en-c ons u ming enzyma tic r eac t ions (Figur e 7D ). HuR KD disrupt s t r anslat ional sig n aling while pr eserving global pr o t ein out put Our DS Gs analy s i s r ev ealed significan t enrichment in pa thw a y s r ela t ed t o tr ansla tional c on tr ol, t here f o r e, we in v est ig at ed sev er al k e y tr ans la tion a l s ignal s. W e asse s s ed the ph os pho ryla t ed s t a tus of the mec hanis tic t ar g et of r a p a m y cin (mT O R), a cen tr al media t or of mRNA t r anslat i on and pr ot ein s yn thesi s . mT OR pho s phor ylat es tw o k e y immedi at e t ar g ets, the p70S6 kina s e ( p70S6K) and euk ary otic initia tio n f act or 4E- bind i n g pr ot ei n 1 (4E- BP1). 35 T ot al le v els of p7 0S 6 K and 4E-B P1 w er e r ed uced in H uR KD c ells. How ev er , their phosphor yl a t ed f or ms, p-p70S6 K and p-4E-B P1 , incr ea sed, r es ult i n g in inc r eas ed phosphoryla t ed- t o -t ot al pr o t ein r atios ( F igur e 8A, B). We ne xt e x amined mi t og en activa t ed pr o t ei n kinas e- i n t er acting serine/thr eonin e kinase 1 ( MAPK- MNK1) a xis, which pr ed ominan t ly r egula t es c ap dependent tr ansla tion init i at ion, the p r edom inan t f o r m of t r ans la t i o n i n e u kar yo tic cells. Ph os ph oryla t ed MN K 1 decr ea sed, wher eas t ot al MN K 1 lev el s r emained u nc hang ed (Figur e 8C). C ons i s t en tly , ups tr eam of MNK1 , the e xtr a cellular signal-r egula t ed kinases1/2 (ERK1 /2) show ed r educed levels o f bo th phosphoryla t ed and t ot a l pr o t ein ( F igur e 8D ). Fur thermore, t hes e pa t hwa y s co n ver g e on the euk ar yot ic initiat i on f act or 4E (eIF4E) , a dir ec t subst r a t e of MNK1 and 4E- BP 1. In H uR KD cells , b oth t ot al and phos ph oryla t ed lev els w er e r educ ed , leading t o a low er p -eIF4E/eIF 4E r at io (Figur e 8 E). T o det er mine whet her th es e mo l ecular c hang es af f ect ed ov er all pr ot e in tr ans la tion , we perf or med a pu r omy cin inco rpor a t ion as sa y t o me a sur e nascen t p r otein s yn thesis r a t e. D es pit e th e pr onounced a lt er ations in these t r a nsl a t ional regula t or s, no s ignific an t diff er ences in global pr ot e in syn t hes i s r a t es wer e det ect ed s u g ge s ting mai n t enance of tr ansla tional ou tput t hrough t he comp ens a t ory ac t iva ti on o f mT O R 1 (Fi gu r e 8F).

R N A f a t e a n d function a r e lar gely det er min ed by its int er action with RBP s which r ecogniz e c ons en su s R NA binding mot if s o r s tructu r al domains t o mo dula te RNA splicin g , st abil it y , loc aliza t i o n and tr anslat ion, known a s pos t-t r ans c r iptional g en e r egulat ion. P os t-tr anscript ional gene r egula tion ult imat ely shape s ph y siological and adapt ive cellular r es ponse t o s tr es s. HuR, i s a ubiquit o us ly e xpr es s ed R BP tha t c ont a ins t hr ee RRM domains that media t e bin ding t o adenine - a n d urid ine-ric h el e men ts ( AREs) .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 22, 2025. ; https://doi.org/10.1101/2025.08.08.668956doi: bioRxiv preprint pr edomina n tly loc a ted within th e 3ZiKU un tr ansla t ed r egions (3ZiKUU TR s ) of t ar g et t r ans c r ipts. Thr ough t hes e in t er actio ns, HuR is best known f or modu la t i n g its t ar g et mRN A st ability and mod ula t ing thei r alt er n a ti v e s plicing 36,37 . H uR has emer g ed as a k e y modu l a t o r of met abolic dis ea se, and its r ole ap pear s t o v ary acr o ss c ells , tis s ues and disea s e mo dels . In ad i p os e t issue, HuR leve ls ar e decr eased in diabet ic and HFD- f ed mic e, and adipos e- s pe cific H uR knock out pr edis p ose s mice t o H FD-indu c ed obesity , insulin r esi s t ance, and g luc o se in t oler ance. 22 In t he liver , H u R e xpr ession is similarly r educed in obesi t y-asso ciat ed s t e a tosis, y et its knoc k out aggr a v a t ed hepa tic s t eat osis in so me mo dels , 38 and in ot her s w or sened insulin r es is t ance and gluco se in t o ler ance. 23 Con v er sely , inc r eas ed HuR e xpr ess ion has been ob s erved in diabetic hu man hear t and r a t r etina, su g g est ing c on t e x t-depend e n t up r egula tion. 39,40 These c on t ext -depe nden t disc r epanci es also manif est in t he sk elet al s ys t em. In ov ariect om y - induced os t eopor os is, H uR leve ls decr e ased , and its ov er e x pr es sion mi t ig at ed bone l o s s, while s ilencing impair ed ost eoblast ic diff e r en ti a tion i n MC3T3-E1 cells . 25 In co n tr as t, a dia b etic mouse model e xhib it ed elevat ed Hu R mRNA l ev e ls, and knoc k down impr o ved gluc ose h omeos t as i s and bone s tru ctur e. 26 S i mil a r l y , h i g h g l u c o se e xposur e incr eased HuR e xpressi o n in MC3T3-E1 cells , wher e silenc in g a t t en u a t e d apop t osi s and enhan ced o st eog enic dif f e r ent ia t ion. 26 C ollec t i vely , t hese r eports un der scor e tha t Hu R r esponds t o met abolic pertu rbations in a tis s u e - a nd cond i t ion- spe cific manner , p os sibly due t o th e ef f ect being enact ed by the t a r g et R N A af f ect e d r a ther t han HuR abund a n c e itself . Our tr ans cript ome wide analy sis in os t eocyt es e x po s ed t o high glucose, r ev ealed mi n i mal diff e r ential g ene e x pr es sion, wher eas alt erna ti v e s plicing c hang es wer e pr ominent, and regula t ed a wide a rr a y of cellular p r ogr ams including E CM pr ot eins, t r anslat ion, and cyt osk elet al or g aniz a t i o n. These f i n d ings pr omp t ed u s to i n v es tig a te po st- tr anscriptional r egula tion un de r high gluco se. T o pr obe t he r egul a t o ry f act or s un derlying t hes e s plicing a lt er a tions, we us ed th e di ff er e n tia lly splic ed g ene s et s t o iden tify RB P mot if enr ic h men t w h i ch iden tif ied s ev er al RBP s including H uR, Z C 3H14 , and HNR N P L a s r ecurr en tly enriched ar ound diff er e n tia lly s p l iced e x on s. H uR emer g ed as a pr ominen t candidat e, app ea r ing i n multip le positions ac r oss v a r ious spl ici n g ev en ts. Ther e f or e, we e x a min ed HuR e xp r e ssion a t bo th the m RNA and pr otein le v el s in os t eocytic ML O - Y4 cells cultu r ed in HG and i n t he bones o f HFD mice. Despit e a signific a n t r eduction in HuR mRN A , p r ot e in levels r emained unchang ed, su g ge s ting po st- tr anscriptional buf f ering. In teres tingly , mannit ol-tr eat ed c ells used t o c ont r ol osmot ic s tr ess e x h i b i t ed a s imilar pa t t e rn , wi t h decr eased HuR mRN A but s t able pr ot ein levels, f urth er i mplicating r egula t o r y mechanisms th at main t ai n pr o t ein .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 22, 2025. ; https://doi.org/10.1101/2025.08.08.668956doi: bioRxiv preprint abun danc e under s tr ess. Pr eviou s s tudies h av e s ho w n tha t H uR pr ot ein level s r emained s t abl e under h ypot o n ic str ess, while its sub cellular loc aliza tion s hif t ed fr o m t he nucleus t o the cyt oplasm. 41 Although we did not dir ec t ly a s se ss HuR localiz at ion in ou r s tud y , it is c onceiv able th at similar st r es s-induced r eloc aliza tion oc cu rr ed in o ur cells , pot en t i ally c on tributing t o the obs erv ed e ff ect on mRNA and p r o t ei n l ev els. Our ob s erva t i o n is al s o co n s i s t en t w it h evidence th at mRNA abundance doe s not r eliably pr edic t pr o tein le v els , due t o tr anslational c ontr ol and b u ff ering mec h a n i sms. 42 G i v e n H uR’ s cen t r al r ole in ma int aining cell ular homeost a sis, its e xpr ession ma y be pr eser v ed t hr ough enhanced tr ans la tion a l e ffici ency or a lt er ed l oc aliza t i o n t o s u st ain functional ou tput. How ev e r , this h ypothe sis r emains spe cula tiv e and w a r r an ts furt her in v es tig a tion usi n g appr oaches su ch as ribosome pr ofiling o r s p at ial pr ot e o mic s. The dynamic s of H uR e x pr es sion under h yper gly cemic s tr es s pr ompt ed us t o in v es tig a t e it s r ole in os t eocyt es. H uR KD in ML O-Y4 c ells e xhibit ed pr o f ound dif f er e n tia l gen e e xp r ess ion , inclu ding r educed e xpr ess ion of g enes in v ol ved in e xtr ac ellular ma t rix r emodeli n g consis t en t with i t s r epor t ed r ole in maint a ining s ev er al E CM pr ot eins including c olla g ens an d ma tri x met al lopr o t ei n as es. 43 DE G s a lso show ed do w n r egula tion in li pi d met abol is m and c yt os k e let al or g a n i z a tion r e f l ec t i n g cellular morphologic al chang es . C ons i s t en t w it h th a t phallo idin s t a ining show ed ro unding and loss of dendrit es aft er H uR KD c onsist en t with G SE A. Enriched pa th wa y s wer e clearly biased t owar ds mRNA quality c on tr ol an d mRN A tr ansla tion. D SGs in HuR K D cell s show ed enrichmen t in ribosomal and mi t ochondr ial t er ms as w ell as t er ms r elat ed to t he tr ansla tional machinery , su c h as rR N A pr oce ssing, RNA spli cin g , and t r ansla tion- dep end en t mRNA sur v eillance pa thw a y s , in cluding NMD , indic a ting an incr eased aberr an t mRNA s and mR NA d eg r ada tion. 44 These finding s highligh t tha t H uR KD induce s s u bs t an tial splici n g a lt er a tions tha t cor relat e we ll with t r ans c r iptional alt er ations. Furt hermor e, AS a n a ly si s r ev ea led s ignific an t enrichmen t in st r ess - r ela t e d pa t hw ay s including aut ophag y and H IF-1α si gnaling a s w ell as k e y met abo lic pa thw a y s su ch a s ins ulin signaling and non- a lc oholic f a t t y l i v er d is ease. Not ably , these pa thwa y s w er e not det ect ed thr ou g h di f f e r ent ial g ene e xpr es sion analy si s , highligh ting AS as a crit ical post -tr an s cript i o nal mechani sm of g ene r egulat ion. These findings empha si ze t he added r esolution pr ovided by spli cing analy s e s, enabling the iden t ificat i o n of r egulat o ry chang e s th at ar e no t appar en t at g ene abundance levels alone. H uR KD incr eased t he sensitivity o f os t eocytic c ells t o gluc ose-dependen t o xidative s tr ess and d ispla y ed elev at ed R OS lev els f oll o w ing e x posur e t o o xida tive ag en ts suppor ting a pr ot ectiv e r ole f or HuR i n maint ain ing r edo x b a l ance. In t e r es tingly , un der baseline cond i t ions , H uR KD led t o a r ed uc t ion in R OS l ev els. TX NI P , is a gl u co se- indu c ible inhib it o r o f th ior ed o x in ( TR X), a R OS sc a v eng er t hat pr o t ec t s ag a ins t diabetic .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 22, 2025. ; https://doi.org/10.1101/2025.08.08.668956doi: bioRxiv preprint bo ne los s. 45 O u r r esults indic a t ed a d ir ec t link betw een TXNI P mRNA and HuR, as H uR c opr ec ipit at ed with TXNI P , and up on H uR d e p l etion TX N IP mR NA lev els and half-l if e incr eased indic at ing t hat Hu R de st ab i liz es T X NI P mRNA. O u r r esults ar e also c or r obor a t ed by pr ev iou s da t a r eporting TXNI P a mong 4874 con sen su s g enes t hat dir ec t ly bind H uR 37 . C oncomit an t wi th th es e r esults, DSG anal y si s show ed an enrichmen t in insulin s ignaling r ela t ed t er ms. T o v a lid a t e we pr obed insuli n r esponsiveness . HuR KD imp air ed insulin signalin g , as evidenced b y r educed AKT phosphoryla tion in r es po nse t o insulin s timula ti o n. Insulin r ep ort ed ly suppr es se s TX N I P e x p r e ssion in ins ulin r esponsi v e cells . 34 Collectiv ely , HuR loss eleva t es TX NI P vi a t w o co or dina t ed mec hanisms, r educed mRN A deca y and a t t enua ted insulin-depend en t tr anscriptional do w nr egula tion, t her eby , inc r eas ing o xida tiv e -st r ess se ns it i vity und e r high glucose. Giv en th at mi t ochondria ar e the p rimary s ou r ce of cell u l ar R OS, w e assessed mi t ochondria l mas s and f un c t ion. HuR KD r edu c ed mit oc h ondrial c on t en t, as e st ima t ed by Mi t otr ac k er s t aining , a n d imp ai r ed mi t ochondr ia l r es pir a t ion, e v iden c ed by decr eased OCR , maximal r es pir at ion, and c oupling e f f ic iency . A TP pr o du c t ion w as also dimi n i shed, i n dica t ing r educed o xidativ e phosphor yl a t ion (OxPho s), while pr ot on leak r emained unchang ed, su g g e st ing tha t m it ochondr ial me mbr a n e i n t egrit y was pr es erv ed. The de cr ease in R OS u nder b a seline c onditions ma y be e xplained by r educed mit oc h ondrial a ct i vi ty and t ot a l o x y g en co ns umption by cells r educing R O S g ener at ing capacity . How ev er , the heigh t en e d v ulner abi lity t o ex og en ous o xida tiv e s tr ess u nder HG and R O S-ind uc ing c onditions highlig h t s a f ailur e of adaptive an t io x idant response i n HuR KD cell s. Collectiv e ly , t hes e f i n dings und er s c o r e Hu R’ s r ole in maint aining c ell v iabili ty under HG c ondit i o ns and mai n t aining r edo x homeos t asis and mi t ochondria l function . Giv en the enrichmen t of AS ev e n ts in pa t hw ay s r ela t ed t o mR N A t r a nsla tion and pr ot ein s yn thesis , we e xamined whe ther these chang es wer e functionally r e f lec t ed in t r anslat ional s ignaling. Euk ar y otic tr a nsla tion c an be initiat ed via tw o mec hanisms, c ap- dep enden t and c ap independe n t t r ans la t i o n. Cap depend en t t r anslation i s the pr edomina n t f o rm and c ap depen de n t tr ans la ti on ini tiation is t he r at e l i mi t ing st ep i n t r anslat ion whic h d epends o n eIF 4 E a v ailabil i t y . 46 eIF4E binds t o the m 7 G c ap loc at ed at t he 5’ end o f mos t mRNAs wher e it r e c r uits other ini t i at ion f act or s pr epar i ng t he mRNA f o r ribosomal loading and scanning f o r the t r ansl a t ion init ia t ion c odon. 46 T h e a c t i v i t y o f eIF4E is r egula t ed via tw o mechanisms. F ir st , it is seques t er ed b y t he inh i bit o r y 4E-BP1 which r el eases eI F4E upon it s p ho s p ho ryla tion by mT OR C1 k ina se. 35 Second , a vailable eIF4E i s pho s p horyla t ed by MNK s which ser v es t o modula t e selec t ive mRN A tr ansla tion. Not ably , phosphoryla tion o f eIF4E b y MN K s is not r equ i r ed f or global mRN A tr ansla ti on, .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 22, 2025. ; https://doi.org/10.1101/2025.08.08.668956doi: bioRxiv preprint c on v er sely , mT O R C 1 inhibition le ads t o near comp l et e inhibi tion of mRNA t r anslat ion. 47,48 This align s well wit h our da t a wher e HuR KD pr eser v ed gl obal pr ot e in ou tput p ossibl y due t o enh anc ed mT OR C1 a ctivity and i n h ibit ion o f 4E-BP 1 which ser v es t o r elease e I F 4E f o r tr ansla tion initi a t i o n. Our da t a r ev ealed r educed t ot al le v el s of p7 0S6 K and 4 E-B P1, y et their p hos ph oryla t ed f o rms w er e eleva t ed, r esulting in i n c r ea sed phospho-t o-tot a l ra t ios —s u g g es ting r elative activ a t ion o f mT O R C1 kinase. In con tr as t, the MN K1- eIF 4E a xis wa s s upp r essed: ph os pho ryla t ed MN K1 and its up str eam kinase ERK1 /2 w er e r educed, and both t ot a l and pho s phor ylat ed e I F 4E lev els w er e diminis h ed. D es p i t e t hes e alt er ations, global pr ot ein s yn thesis r a t es r ema ined u nc h a n ged . T h e pr eser v a tion of global pr ot ein outp ut despit e disrupt ed s ignaling in k ey t r a nslat ional pathw a y s su g g e st s a s h i f t tow ar d b i ased mRN A t r anslat ion, w h er e the ce ll p r iori tiz es th e s yn thesis of spec ific mRNAs. Su ch r epr ogr amming ma y r epr esen t an a d a p tive response t o pr es erv e pr ot e os t as i s u nder ener g etic and met abolic s tr es s. Alt hough we did n ot meas ur e selec t ive mRNA t r anslat ion i t is pla u s ible that a s hift tow ar ds incr eas ed tr ansla tion of mR N A s r elat ed t o cell s urvival has t a k e n p l ace in HuR KD cells . In conclu sion, our st ud y eluci d at es t h e r ole of Hu R in ost eocy t e biology , particularly in r egulat ing AS, t r ansla tion, mit oc h ondrial fu nc t ion and r edo x homeo st asis . HuR s eems t o pla y a pr o tectiv e r ol e ag ainst high glucose a n d o xida tiv e cell d e at h. O ur find i n g s r eveal t hat H uR is indis pensable f or main t a ining cellular and met abolic homeos t as is in os t eocyt es, p a r ticularly u nder HG c ondit i o ns . Our da t a pr ovides a mechanistic un der s t anding of H uR’ s r ole in cellular homeos t asi s and ha s b r oader implic at ions in met abolic bo ne dis eases .

1. P rima r y cells an d c ell lines Pr imary os t eocyt es w er e isolat ed fr om n eon a t al c al v ar i ae of C57B L /6-T g (D mp1- T opaz)1I k al/J u s ing s erial enzyma tic d i g es tion w it h c ollag enas e a n d ED T A f ollowed by f l u or esc en t activa t ed cel l s or ting (F ACS) a s de s cribed pr evi o us ly . 49 A f t e r i s o l a t i o n , c e l l s w er e cultur ed a t s t andar d c ondition s (37 /i1ZiKU, 5% C O 2 ) o v e r n i g h t in a l ph a - m in im um es se n tial mediu m (ZiKU-MEM; Wa k o Pu r e Chemical In dust ries, O s a k a, Jap a n ) c on t aining 10% f et al bovin e s er um ( FBS; Biowes t, Nuai llé, Fr an ce), 100IU/mL p enic illin G , 100 μ g / mL str ept omy cin ( 1% P / S) c on t ai ning 5.5 mM gluc o se ( s t andar d medium). Aft er t ha t, th e med ia w as r eplac ed w it h fr esh 5.5mM gluc o s e ( normal gluc ose, NG), 2 5 mM gluc ose (Si gma-Aldr i ch, St . L ou i s, MO , USA) (h i gh g luc o se, HG) c on t aining media or 5.5 mM gluc os e supplemen t ed wi t h 1 9.5 mM mannit o l ( os mo tic con t r o l, M) (Sigma- A ldrich) f o r 3 d ay s. ML O-Y4 cells (Adde xBio T echnologies ) w er e ma int ain ed a t 37 /i1ZiKU, .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 22, 2025. ; https://doi.org/10.1101/2025.08.08.668956doi: bioRxiv preprint 5% C O 2 in s t andar d medium and k ept a t 70-80% conf luenc y a n d passa g ed as needed. St and ar d medium w as chan g ed t o NG , H G o r M media f or s ubsequen t e xper i ments. 2. Mice an d f ee ding e xperime n ts 12- week-o l d male C57BL/6J mice wer e pur c h a sed fr om C L E A Japan (T o ky o , Japan). Mic e w er e accl imat ed f or 4 w eek s in specific pa thog en-f r e e (SPF) co ndit ions under a 12- h li gh t / dar k cy cle wit h a d l ibit u m f eeding (R esear ch Diet, D 12450H , J apan) and a t 16 w eek s the mice w er e r andomly alloc a t ed t o a 10% K cal fr om f a t t ermed normal diet ( N D ) ( R e s e a r c h D i e t , D 1 2 4 50 H , J a p a n ) o r 4 5% K c a l f r o m f a t t e r m e d h i g h f a t d i e t ( H F D ) ( R es ear ch Diet , D 12451 , Japan) f or 16 w eek s t o induce chr on ic h yper gl y c emi a. The mi ce w er e t est ed f or f ast ing blood gl uc ose and gluc ose t oler ance a t 4 and 8 w eek s of diet a n d on e da y b ef ore c ollec t i n g t h e bones at 16 week s of diet with 5 mi ce per gr oup. A f t er s acrif i ce, t he long bo nes (f emu r , t ibia, hume r us ) w er e e x ci sed. The musc le s and soft tis s ues w er e car e f ull y r emov ed, and the bo nes wer e scr a p ed usi n g a sc alpel. The epiph y se s w er e cut and th e bone marr ow flus hed in phospha t e- buff e r ed sa line (PBS) x1 a t 9000 x g , 4 /i1ZiKU thr e e times . The bones w er e immedi a tely fr o z en in liq uid nitr og en and st or ed a t -8 0 /i1ZiKU f or RN A and pr ot ein e x t r action. A ll procedur es in v olvi n g a n i mal s w er e perfor med in a cc or dance with the ARRIVE g u i d el ines and wer e app r ov ed by the R egula t i o ns f or Anima l Ex per iment s and R ela t ed Activit ies a t T ohok u U niv er sity ( 2021DnLMO-0 07- 04). 3. B lood glucos e m eas ur emen t and glu c ose t oler ance t est F as ting bloo d gluc o s eand glu c os e t oler a n c e w er e as sessed aft er a 6-ho u r f ast ( 8:00 AM–2:00 PM ) u sing a r oden t sp eci f ic glucomet er with comp at ibl e L G s en s or s ( L abGluc o Funa k o s hi., T o ky o , J apan). Ap pr o ximat ely , 5 μ L or les s of blood w as dr a wn f r om a t a il t ip clip at 0, 15, 30, 60, and 90 minut es. 4. Sho rt hair pin RNA knock dow n T w o shor t hair pin RNA (s h R N A ) seq uences t ar g eting mouse HuR / ELA VL1 mRN A w er e designed us in g V ect orB uilder ’ s sh RNA T ar get D es ign t ool. An emp t y bac kbone sequence w a s u sed a s a non-t ar get ing con tr ol. The shRNA oligonucle otides w er e or der ed and s yn thesi zed b y F ASMA C Co., L t d. (K anag aw a, J apan). Oli go s w er e annealed and lig at ed and cl o ned int o pLK O.1 pur o v ec t o r ( Addg ene, Plas mid# 8453). Plas mids w er e then e x t r act ed u s ing Nu cleos p in plas mi d DNA purific a tion k it (Mac her ey-N ag el, D ür en, German y) f ollow ed by v alidating the ins er ti ons u s ing co l o n y - PCR con firming sequence s b y Sang er Sequenci n g (Eur ofins Genomics , T ok y o , J apan). HEK293 T cells w er e c o-t r ansf ec t ed with shRNA plas mids , len tivir al packagin g plasmid psP AX2 ( Addg ene, Plasmid#12260) and V SV - G en v elope e xpr es s ing pla smid pMD2 . G ( Addg ene, Plasmid#12259) using L ipof ect amine 3000 (Ther mo Fis her Scien tific, Waltham, MA, .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 22, 2025. ; https://doi.org/10.1101/2025.08.08.668956doi: bioRxiv preprint USA). V ir al part i cles w er e c oll ect ed a ft er 48 and 72 hour s , cen tr ifug ed a t 1500 x g f or 5 mi n ut es and super nat an t w a s fil t ere d thr ough a 0.45 µm PVD F filter . V i r al par ticl es w e r e s t o c k e d a t 4 /i1ZiKU f or imm ed i at e us e or -80 /i1ZiKU f o r l ong t erm s t or ag e. ML O-Y 4 cells w er e gr own t o 80% conf luenc y in s t andar d medium , t hen media w as chang ed t o t r ansdu ction medium c on t a ining st andar d medium a n d lentivir al par t i cles c on t aining super nat an t a t 4:1 (by volume) and 8 µg / mL poly b r ene (Sigma-A ldrich, S t. Louis, M O , USA) f or 24 hour s . T r an sdu ction medium w as then r eplaced with s t andar d medium and incuba t ed f or 6 h our s th en pur omy cin w a s ad ded a t (2 µ g / mL) and t r an sf ect ed cells w er e select ed f or 7 da ys be f or e passa gi n g . C on st ruct sequence s of con t r ol Mo c k knoc k down (Mock KD ), c ons tr uc t A knock do wn (Y A KD ) and c ons tru c t B knock do w n (YB KD ) in S u pplement ary T able 5. A and B d enot e 2 s epar a t e c onst ructs and ( Y) denot es t hat MLO-Y 4 cell s wer e used f or knoc k do wn. 5. R N A s equ e nc ing T o ta l R N A wa s ex t rac te d u s i n g Q i a g e n RN e as y k i t (Q I A G EN, Hi l d e n, G e rm a ny ) w i th a D N a se dig es tion s t ep ( Q IA G E N) as p er manuf actur e r ’ s inst ructions. The s amples w er e pr ocessed b y Azen t a, Japan as f ollow s : s amples w er e scr eened and qu a n tif ied o n an A gi len t 4200 T apest at ion wi th R N A S cr e en T ape. The r es u l t an t RIN (R NA in t egrity nu mber) w as 9. 9 or 10 f or al l s a mples . mR NA w a s enriched u s ing N EB Ne xt P oly ( A) mRN A Magnet ic Isola tion Module (NEB) and R N A libr ar y pr epar at ion w as done NEB Ne x t U ltr a II Dir ectional RNA L ibr ar y Pr ep Kit (Il lumina E7760) . Sa mples w er e po oled on an Ill u m ina N ov aseq t o obtain pair ed en d 150bp r eads w it h a t arg et dept h of 50 mil lion r eads p er sa mple. F A S T Q files wer e then used f or bi oinf or matics analy s i s. All sequencin g e x per imen ts wer e don e i n tr ipl ica t es. 6. R N A s equ e nc ing a naly sis Quality co n tr ol of r aw F A S T Q files w as c onduct ed us in g F a stQC. Adapt er s and low- qu a lit y r eads w er e r emoved us ing T r immomatic. 50 T rimmed r eads wer e ali gned t o the mous e r e f erence g enome (M35, GR C m39) usin g the splice-aw ar e aligner HISA T2. 51 T h e r es ult i n g B AM fil es of mapped r ea ds w er e qu a n t i fied ag ai n st g ene f e a t ur es us ing F ea tur eCoun ts wit h de f au lt par amet er s. 52 Di f f er ential ly e xpr ess ed g enes (DE G s ) w er e iden tified us ing Limma-V oom, 53 app lying TMM no rmaliza t i o n an d d ef in i ng D E Gs a s t hos e with |log₂ f old c hang e| ≥ 1 and adjus t ed p value < 0.05. 7. A S and RBP m otif analysis A li gned BA M files w er e used t o c o nduct AS analy si s u sing t he rMA T s-t ur bo suit e. 32 St a t istic al s ignific ance of AS ev en ts was c ons ider ed a t FDR 0.2 t o f i lt er in diff er e n t ia l ly spliced g ene s (DS G s ) . Spli c e motif anal y sis wa s c on duct ed us in g rM A Ps 2 . 33 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 22, 2025. ; https://doi.org/10.1101/2025.08.08.668956doi: bioRxiv preprint 8. Gen e on t ology and fu nction al e nrichmen t analys is D SGs w er e us ed f or functional enric hmen t analy s es . The output o f rMA T s w a s piped in t o r M AP s t o g ener a te RBP mo tif maps. 33 D E Gs a n d D S Gs w er e analy zed f or g ene on t o logy ( GO) and f unctional enrich men t analy s is u sing ei t her Da vid G O functional anno t a t ion t ools or eV IT T A ’ s pr e-r ank ed g ene set enr ic h men t analy si s ( G S E A) or ov er r ep r esen t ation analy s is ( O R A) toolbo x. 54 Figur es in this pu bli c a tio n wer e made using be spo k e s cripts in Rst udio 4.4. 0 ggplot or g gplot 2 pack ag e s and e xt ensions . 9. Cell viabilit y assa y In a 96- well pla t e, 1×10 4 M o c k KD , Y A KD o r Y B KD w er e seeded in eac h w ell . Cells w er e cultu r ed in each w el l with 200 μ L of α- MEM supplemen t ed wi th 10% FBS an d 1% P / S. C e ll viabili ty wa s mea sur ed a t 0, 2 4, 4 8, and 72 ho ur s. Th e plat e was then i ncuba t ed f or 2 h our s a t 37°C wit h eac h w el l c ont ai ning 100 µ L of α-MEM and 10 µL of C el l C oun ting Kit -8 solution ( Doj indo Labor a t or ies, K umamot o , Japan). Absorbance w a s meas ur ed a t 450 nm us ing a mi cr opla t e reader (Su nrise™ R emot e, T ecan, Männedorf , S witz erland). 10. R eal-t ime polymer as e chain r eactio n (qPCR ) R N ea s y kit (QIA GE N ) w a s u sed t o e x t r act t ot al RNA fr om cells a cc or ding t o ma n uf ac t ur er ' s ins tru ctions and T ri z ol-chlor of or m w as u sed t o e xtr act R N A f r om bones. 1 f emur w as immer sed i n 1 mL T ri z ol and pulv eriz e d 5 times at 5000 R PM s peed set ting f o r 60 se co nds each in T OMY Micr o Smas h MS 100 Cell Disrupt or . T ot al RNA w a s u sed t o s y n t h e s i z e c D NA u s i n g t h e S u p e r S c r i p t ® I V F i r s t - S t r a n d S y n t h e s i s S y s t e m ( I n v i t r o g e n , C a r ls bad, CA, USA) acc or ding t o man u f ac t ur er's ins tr uc t ions . mR NA e xpr e s s ion v a lues wer e mea sur ed u s ing the CF X96 T ou c h R eal-Time PCR D et ection S ys t em ( B i o-Rad). 11. RNA Im munopr ecipitation (RIP ) assa y R IP ass a y wa s p erfor med us ing t he EZ- Magna RIP kit (Mil lipor e, Bi ller ica, MA) ac cording to t he manufactur er’s i n s t ructions. Briefly, cells at 80% confluenc y w er e s craped off and lysed in RIP ly s i s buffer p rovided in the kit. Then 100ZiKUµ L of whole cell lys ates wer e incubat ed wi t h RIP buff er containing magnetic beads conjugat ed with ant i- HuR ant ib ody (or n ormal IgG as negative c ont rol, Anti-SN R NP70 a s po s it i ve cont rol). After washing, R N A –protein comple xes were eluted and digested with pr oteinase K, and the RNA was pur ified usi n g t he ki t reagents. Purified RNA was subje cted to cD N A synthe sis and q PC R anal ysi s a s de s cribed in t he RT-qPC R section. 12. m RNA st ability ass a y .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 22, 2025. ; https://doi.org/10.1101/2025.08.08.668956doi: bioRxiv preprint T o est ima t e mRNA hal f- lif e, nasc en t tr anscription w as bloc k ed with Actinomy cin D ( ActD , final 10ZiKUµg /ZiKUmL ) . A t t ZiKU=ZiKU0, 1 , 2, 4, 6, 8 ZiKUh aft er addi t ion, cells w er e r apidly w ashed w it h ice-c old PBS and ly s ed in TRIzol and R T-qPCR w as done a s des cribed. mRN A d e c a y r a t e w as mea sur ed by non-linear r eg r es sion cur v e fit t ing (on e ph a se dec a y) u si n g Gr aph P ad Pr i sm u sing t he f ollowing par amet er s: goodnes s of f it w as qu a n tif ied with R 2 , u s ing or dinar y fit a t c onfidence level 95%. 13. In s ulin r es po nse t est s Mock KD o r Y B KD c ells w er e seeded in α-ME M supplemen t ed wi t h 10% FBS and 1% P / S un t il r eaching 80% c onfluenc y . The media w e r e t h en r ep laced with α-MEM c on t aining 1% P /S and 1% F B S f or 3 hour s f o l low ed a swit ch t o the s ame media wit h or witho ut 100 nM insulin (Sigma-Aldr ich). A f t er a 30-mi n ut e incuba ti on pe riod, c ells w er e harv est ed and pr ot ei n e xtr act ed using r adioimmunopr ec ipit a t ion (RIP A) assa y buf f er ( Millip or e, Bur l ing t on, MA, US A ) co n t aining 1% pr ot eas e and pho spha t ase inhibit or c oc kt ail (Thermo Fis her S c ien t ific ) on ice f or 20 minut es; in soluble ma t e r ial w a s separ a t ed by cen tr i f ug at ion a t 14000 xg. T ot al p r ot ein w as c ollec t ed f or w es t ern blo t analy sis. 14. R eactiv e o xy g en specie s d et ec tion ass a y Mock KD o r Y B KD ( 2 . 5 × 1 0 4 c ells per w ell) wer e seeded in a black, cl ear bott om 96-well mi cr o plat e overnigh t. In t r acellula r r e activ e o xy gen s pe cies ( R O S) lev els wer e measur ed using t he D CFD A/H2D CFD A- Cellular R OS A ss a y Kit ( Abc am, C amb ridg e, U K) ac c or ding t o manuf a ct ur er's ins tru c t ions . Cells wer e s t ained with 20 μ M D CFD A in H BSS f or 45 mi n ut es a t 37°C . Moc k KD o r Y B KD cells t r ea t ed wit h 50µM TBHP f or 4 hou r s w er e u sed as po si t ive c ontr ol. DCF fluo r es cen ce in t ensity was measur ed u sing a f l u or e s c ence pla t e r eader (Fle x St ation 3, Molec u l ar Devices, San Jo se, CA, USA) with e xcit a tion a t 485 nm and emi ssion a t 535 nm. 15. W e st ern blo t C e lls w er e ly s ed us ing R IP A assa y buf f er ( Mil lipore, B url ing t on) con t ai ning 1% pr ot ea se and phospha t ase inhibi t or cockt ail ( Thermo Fisher S cien tif i c) on ic e f or 20 minut es; insoluble ma t e rial w as s epar at ed b y cen tr i f ug a tion 14000 x g. Pr ot ein fr o m bones w a s e xtr ac t ed in RIP A bu f f er and pr ocess ed simila r t o RNA e xtr action abov e. T ot al pr o t ei n w as quan tified usi n g Pier c e™ BCA p r ot ein ass a y k it ( Thermo Fisher Sc ien t i fic ) . T o pr epar e f or SDS-P AGE g el electroph or esi s, t he pr ot ei n w a s tr eated with β- Mer c apt oeth a n ol ( Bio - Rad Labor at ories) and L aemml i sample bu f f er ( Bi oRad Labor a t or ies) 3:1 and d enat ur ed a t 95° C f or 5 minut es. Eq ual pr ot ein a moun ts w er e loaded o n t o 4–15% Mi n i - PR O TE A N T GX P r eca s t G el s (Bio-R ad Labora t ories ) and t r ans f err ed t o a T r ans-Blot T u rbo T r ans f er S y st em (Bio- Rad Labor at orie s ) a n d then .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 22, 2025. ; https://doi.org/10.1101/2025.08.08.668956doi: bioRxiv preprint block ed with Block-Ace (D S Phar ma Biomed ic al, Osak a , J apan) 1 ho ur a t r oom t emper atu r e (R T). Prima r y a n t ib o dies w er e incuba ted ov e r nig h t at 4° C. The membr anes wer e inc u bat ed with hor s er ad i sh per o x idase-c onju g a t ed an ti -r abbi t an t ibody (Cell Signali n g T e chnology , Dan v er s, MA , USA) at a dilution o f 1:5,000 or an ti- mous e an t i b ody ( G E H ealthc ar e, Chic ag o , I L , U S A ) a t a dilution of 1:10,000 f or 1 hou r a t R T aft er being w ashed in tris-buf f er ed saline with T rit on X-100 ( TBS-T). Bound an t ibodi es wer e det ect ed with SuperSignal Wes t F emt o Ma xi mum Sensi t iv i t y Sub st r a t e ( Thermo Fisher Scien tific) and a FU S I O N -F X6 ED G E Chemi lumi n es cence Imaging S y s t em ( Vilber Lou rma t , Coll égie n , F r anc e) . P rimary an tib od i es ar e in Supple ment a ry T able 5. 16. Co nf ocal micr osc op y 1 × 10 5 M o c k KD , Y A KD o r Y B KD cel ls w er e seeded in 35mm glas s b ot t om di s h over nigh t. C e lls w er e fix ed wi th 4% f ormaldeh y de f or 1 5 minu t es, t hen w ashed 3 t imes with x1 PB S . C el ls w er e permeabi li z ed using 0 .1% T r it on X-100 f o r 10 min ut e s and block ed with 1.5% bovine serum albumin ( BSA) f or 30 minut es at R T . Cell s w er e incuba t e d with A l e x a Fluor 568 Phalloidin ( In vi t r og en) f or 1 hour at R T . Aft e rwar ds, cell nuclei wer e s t aine d with 4’ ,6-diamidino -2-ph en ylindole, dih y dr ochlor ide (D API ) (Thermo F isher Scien tif ic) f o r 5 mi n ut es a t R T . F or mi t ochondr i al st aining , 5 × 1 04 liv e cel ls wer e incuba t ed with t h e M i t o T r a c k e r ™ R e d C M X R o s F M ( I n v i t r o g e n ) i n a g l a s s b o t t o m d i s h f o r 4 5 m i n u t e s . C el l s w e r e w a sh e d wi t h x1 P B S 3 t i me s an d f i x e d wi t h 4 % f o r ma l de h y d e f o r 1 5 mi n u t e s t hen w as hed b y x1 PB S. Cells w er e incuba t ed f or 10 min ut es in x1 PBS c ont aining 0.1% T rit on X-100 and s t ained with D API f or 5 minut es a t R T . Con f ocal imag es w er e acquir ed using a Zei s s LSM800 c onf o c al las er sc anning mic r osc ope (Car l Zeis s , Oberk o chen , German y). Conf oc al micr ogr aph s a c q u i r ed with 10× and 20× objectiv es f or phalloid in s t aining , and 40x f or Mit oT r ac k er ™ a s a s ingle optic al s lice. 17. Seaho r se m it ochondr ial mit o- str ess t es t Ox y gen c onsumpt i o n r a t e (OCR) w as mea s ur ed usi n g t he Seahor se X F96 a n al y ser ( Agilent T echnologies, San t a C l ar a, C A, U SA) an d Mit o -s tr es s t est k it ( Agilent T echnologie s ) along with S eaho r s e XF e96/XF Pr o Flux P ak (Agilen t T ec h nologies ) and Seahor s e XF D MEM medium ( wi t hou t Phenol R ed/pH 7.4/ with HEPE S/ 500 mL , Agilen t T echnologie s ) . Initially , Moc k KD and YB KD cell s w er e s eed ed a t 25,000 cells/well in 96- w ell plat es on da y one, while the XF e 96 sens or c ar tridg e h y dr at ed in 200 µ L c a libr a tio n medium. On the ne xt da y , the cell m edium w as r eplaced wi t h Seahor se X F DMEM, and cells wer e inc u ba t ed a t 37 ℃ in a C O₂ -f r ee incuba t or f or 1 h ou r . Dur ing thi s time, 1 µ M oligomy cin, 0. 5 µM F C CP , an timy cin A, and r ot enone w er e loaded int o t he drug por ts of t he c artr idge. Af t er loading , t he sensor plat e w as c a libr a ted in t he analy s er . F ollowing c alibr a tion, the cell c ult ur e pla t e was l o a d ed, and the analy s i s initia t ed with the .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 22, 2025. ; https://doi.org/10.1101/2025.08.08.668956doi: bioRxiv preprint pr ogr am: s et : Mixtur e 2 minut es, Meas ur e 3 minut es, 4 set s in all s t ep s. P o rts : (A: Oligom y c in / B: F CC P / C : An timy cin/ R ot enone). 18. P ur om y cin incorpo r a tion assay Na scen t pr ot ei n syn the sis wa s ev alua t ed by pur omy c in inc or por a tion as sa y . Cells w er e t r eat ed with 10 μg /mL pur omy cin (Si gma-A l d rich) f or 30 minut es pr ior t o har v es t. Cell ly sa t es wer e pr epar ed and analy s ed by west ern blot as pr eviously d escribe d. 19. St a tist ic al analysis A ll e x pe r ime n ts w er e done in 3 or m or e r ep lica t es . St at istic al analy s is w a s perf or med using G r aph P ad Pri sm 9.0. Analy s is of two gr ou p compa ris o ns was d one us i ng s tuden t ’ s t t est and mult iple gr oup c omparison was d one using o ne-w a y ANO V A f oll o wed by T uk e y ’ s pos t ho c t es t. p v a lues of less th a n 0 .05 wer e consider ed signific a n t .

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Figure legends Figur e 1: H igh glucos e induces minimal dif f e r en t ial g ene e xpr ess io n and e xt ensiv e AS chang e s in prima r y ost eocyt es. (A ) The V o l c ano plot comparing the log2 F C and -log10 adju s t ed P v alue of RN A-seq da t a in prima ry os t eocyt es with g enes s h owi n g t he hig h est chang e s l abelled. (B ) The par en t p ie chart s h ows s ignific an t AS ev ent s (2%) of t he t o t al ev ent s a t t ar g et and jun c t ion r eads (JC E C) and t he s ec ondar y pie chart show s the per c en t ag e o f signific a n t AS even t s of each type (SE, A5SS , A 3SS, MX E, R I) fr om t o tal signific an t ev en ts in pri mar y ost eo c yt es. .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 22, 2025. ; https://doi.org/10.1101/2025.08.08.668956doi: bioRxiv preprint (C - G ) V olcan o plots a n d over r ep r es en t at i on analysis of DS G s ar o und AS ev en ts s ho wing inclusion lev e l ( ΔZiKU) dif f e r ence comparing 5.5mM and 25mM gl u co se a t FD R < 0.05 and 0.2 < ΔZiKU < -0.2 in prima r y os t eocyt es . O v er r epr esen t ation analysis w a s d one thr ough t he eVIT T A web-based t oolbo x. A l l a n a l y s is w a s d o n e o n n = 3 . A ls o , see S u pplemen t ary T able 6. Figur e 2: RBP s motif s significan tly enriched ar ound A S ev en t s. ( A-B ) Th e x a x is (A tt ribu t e) r epresen t s the p os ition (R) of ea ch splicing eve n t i n 5’ → 3’ . R BP binding mot if s a r e desc r ibed in th e y a x is. The panels s h o w SE R BP mot i f s ar ound up and d own r egula t ed DSGs and bu b ble siz e and c olor d enot e the – log10 P v alue. ( C) Example p os ition a l dis tr ibut ion m ap of the HuR b inding mot if a r ound tar get S E e x on. P o sitional dis tribution maps wer e gener a t ed using r MA P s 2. In t r onic r egions ar e 250-bp f l anking sequen ces, and e x on r egions ar e 50-bp sequence s fr om the s t art o r end of t h e e x on wher e th e RBP binds a r ound th e spliced e x on ( g r een) and c onst ituti v e e x ons ( g r ey). The s olid r ed and b l u e l ines ar e the mo tif en r i chmen t s c or es, and the dot ted lines ar e the p v alu es in up and d ow n en riched moti f s, r espe ctiv el y . Black li ne r ef er s t o bac kgr ound R BP s dis tr i b ution. A ls o , see S u pplemen t ary T able 7. Figur e 3: H uR m RNA is r educed in ML O-Y4 c ell s cultur ed in H G an d in th e lo ng bone s o f H F D f ed mice, while main t aining HuR pr ot ein lev els. (A ) H uR mR N A le v el in MLO-Y4 cells und er n ormal gluc os e ( NG), hi gh gluc ose (HG) and osmotic c on t r ol ma n nit o l ( M). Da t a ar e p r es en t ed as mean ± S D , n=4. *p<0.05, ****p<0.000 1. (B ) H uR pr ot ein le vel in MLO-Y4 cell s in n or mal gl u cose (N G ) , h i gh gluc ose (H G ) and osmot ic c on tr ol manni t ol (M). Dat a ar e p r es en t ed as mean ± SD , n=7. (C ) H uR mR N A lev el in the long bones of high f at diet (HFD) and normal diet (N D ) f ed mi ce. D at a ar e presen t ed as mean ± S D , n=4. *p<0.05. (D ) H uR pr o t ein l ev e l in the long bones of high f a t (H F ) and no rmal diet (ND) f ed mi ce. D at a ar e p r esen t ed as mea n ± S D , n=4. *p<0.05. Figur e 4: H uR K D co nfir ms f unctionalit y in in ML O - Y4 cells. (A ) W e st ern blot analy s is of HuR pr o t ei n lev els a f t er s hR N A- mediat ed knock down in MLO-Y4 cells . D at a a r e pr esen t ed a s mean ± SD , n =3. ** p <0.01. .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 22, 2025. ; https://doi.org/10.1101/2025.08.08.668956doi: bioRxiv preprint (B ) C CK c ell viabili t y a ss a y . Da t a ar e p r ese n t ed as mean ± SD , n=4. **p<0.01 . (C ) C onf oc al micr os copic imag es of phall o i d i n st ai n i n g , Scale b a r s : 200 µm f or the 10× imag e and 100 µm f or the 20× im ag e. (D ) C y t oplas m t o nucleus r at io. The m ean of 5 analy sed r egions (co rner s and cen t er ) o f a 35 mm glass bot t om dish wer e us ed. D a t a ar e p r esen t ed as mean ± SD , n=4. ****p<0.000 1. (E ) V o l c ano plot co mparing the l o g2 F C a n d -log10 adju s t ed P v alue of RN A-seq d a ta ( D E G s ) a f te r Y B KD in ML O-Y4 c ells , r ed arr ow =H u R. n=3. (F ) Gene set enr i chmen t analy s is u s ing e V ITT A t oolbo x af t er YB KD o f D E G s a t L o g F C 1 and - l o g 10 adju st ed p<0.05. Figur e 5: H uR K D induc es AS ch ang e s in ML O-Y 4 cells (A ) The par en t p ie chart s h ows s ignific an t AS ev ent s (3%) of t he t o t al ev ent s a t t ar g et and junction r eads (J CE C) and second a r y pie char t sho wing t he p er cen t a g e of signific a n t AS even t s of each type (SE, A5SS , A 3SS, MX E, R I) fr om t o tal s ignific an t ev en ts. (B ) Sc a t t er pl ot showing D S Gs ar ound A S ev en ts in 5 pa tt er ns FD R < 0.05 a n d 0.2 < Δ ZiKU < - 0.2. (C ) Gene On t o l o g y Cellular C omponent (GOC C) en richmen t analysis of DSG s per f ormed us ing th e D A VID F u nc t ion al Anno t a tion T oo l . (D ) ORA of DSGs aft er YB KD . A ls o , see S u pplemen t ary T able 8. Figur e 6: H uR kno c k dow n h eig h t en s o xid a tiv e st r ess th r ough e nh anc in g TXN IP s t abilit y . (A ) C e ll viabil i t y as sa y i n YB KD and Moc k KD cells in 5.5mM gluco se ( NG), 25 mM gluc ose (HG), mannit o l ( M) and H 2 O 2 s t i mu l a t e d c e l l s. D a t a a r e p r e s e n t e d a s me a n ± S D , n=4. *p<0 .05, ** p <0.01,***p<0.00 1, ****p<0.0001. (B ) R eactiv e o xy g en species (R O S ) measur eme n t. F old chang e is c alc u l a t ed aft e r t r ea t ing cells wit h TBHP t o induce R OS f orma ti on. F old chang e r epr es en t s TB HP s timula ti o n/ bas al line c ult ur e c onditi o ns . Da t a ar e pr esen t ed as mean ± SD (n=5 per gr oup; *p<0.05, ***p<0.001) (C ) TX NI P mRNA lev el in MLO-Y4 cell s af t er H uR KD . D a t a a r e presen t ed as m ean ± SD , n =3. * *p <0.01. .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 22, 2025. ; https://doi.org/10.1101/2025.08.08.668956doi: bioRxiv preprint (D ) H uR RIP -qPCR in ML O-Y 4 cells demons tr a t ing TXNI P mRNA enrichmen t in H u R immunoprecipit at es r el a ti v e t o IgG co n tr ol. D a t a ar e pr es en t ed as r elat iv e qu a n ti ty no rmalized t o in put pr ep r esen t as mea n ± SD , n = 3 , ****p < .00001. ( E ) TXNI P mR N A st a b i lit y as sa y f ollowing actinomy cin D tr ea tment. D ec ay c urv es w er e fi t t e d using a one-p has e e x po nen tial mo del; hal f- l i f e incr eased f r om 1.47 h in Mock KD t o 1.85 h in YB KD . D at a poi n ts r epr es en t mean ± SEM (n = 6) , p < 0.03 f or deca y r a t e con st an t (K) c omparison. (F ) A KT / a ctin and p-AK T / AKT pr o t ein lev els in YB KD and Mock KD c ells. Da t a are pr es en t ed as mean ± SD , n=4. *p<0.05. Figur e 7: H uR kno c k dow n in duce s mit oc h ondrial dys funct i o n (A ) Fluor es cen t micr osco pic imag es of Mi t o tr ack er CMXR os R ed s t aining mi t ochondria. (B ) Me a n f l u o r e s c e nt i n t e ns i t y (M F I ) o f Mi to tra c ke r C M XRos Re d st a i n i n g mi t ochondria. D a t a a r e pr esen t ed as mean ± SD , n=4. ***p < 0 .001. (C ) Seahor s e XF Cell Mi t o-St r es s show ing o xy gen c on s umption r a t e (OCR) in pmol/ min. (D ) P ar amet er s of mit ochon drial r espir a ti on. D a t a ar e pr esen t ed as mean ± SD , n=4. *p<0.0 5, **p <0.01) Figur e 8: H uR kno c k dow n in duce s tr ans la t ion dysr egula tion a n d pr es er v es global p r ot ein s yn the s is r a t e (A ) W e st ern blot o f p-p70S6K and p70S6 K pr ot ein l evels. n=4. (B ) W e st ern blot o f p-4E-BP1 and 4E-B P1 pr ot ein lev e ls. n =4. (C ) W e st ern blot o f p-MNK1 and MN K1 p r ot ein leve ls. n=7. (D ) W e st ern blot o f p-ERK1/2 a n d ERK1/ 2 pr ot ein lev e ls . n=4. (E ) W e st ern blot o f p-eIF4E a n d eIF 4E pr ot ein lev els . n=4. (F ) W e st ern blot o f pur omy cin incor por a tion as s a y . n=4. D a t a ar e p r esen t ed as mean ± S D of 2 or mor e independe n t b lots ( *p<0.05, **p<0.01 , ****p<0.000 1) .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 22, 2025. ; https://doi.org/10.1101/2025.08.08.668956doi: bioRxiv preprint Sup plemen t ar y Fig 1: High g lu c ose induce s e xt en siv e A S chang es in p r i m a ry os te oc y t e s . (A - C ) V olcano p l ots a n d over r ep r es ent at i on analysis of DS G s ar o und AS ev e n ts s howin g inclusion lev e l ( ΔZiKU) dif f e r ence comparing 5.5mM and 25mM gl u co se a t FD R < 0.05 and 0.2 < ΔZiKU < -0.2 in prima r y os t eocyt es . O v er r epr esen t ation analysis w a s d one thr ough t he eVIT T A web-based t oolbo x. Al l an alyse s wer e d one on n=3. Sup plemen t ar y F ig 2: Significan t ly u p -enriched RB P s m otif s a r oun d AS ev e n ts. (A - D ) The x a x is (A t tr ibut e) r epr es en ts t he po si t ion (R) o f eac h s plicin g even t in 5’ → 3 ’ . R BP binding motif s are desc r ibed in the y a x is. The panels show MXE , A5SS, A3SS and R I RBP motif s ar oun d up and down r egula t e d DSGs a n d bubble si z e and c olor d eno t e t he – l o g 10 P v alue. Sup plemen t ar y F ig 3: Significan t ly d o wn-enr ic hed R BP s motif s ar ou nd AS ev en ts . (A - D ) The x a x is (A t tr ibut e) r epr es en ts t he po si t ion (R) o f eac h s plicin g even t in 5’ → 3 ’ . R BP binding motif s are desc r ibed in the y a x is. The pan e ls show MX E, A5SS, A3SS and R I RBP motif s ar oun d up and down r egula t e d DSGs a n d bubble si z e and c olor d eno t e t he – l o g 10 P v alue. Sup plemen t ar y Fig 4: W eigh t and met abolic t est s of m i ce f ed a high f a t diet f or 16 w eek s. (A ) Six-hour f ast ed b lood g luc ose lev el s of mice f ed a ND o r H F D . Dat a ar e pr es en t ed as mean ± SD , n=5. (B ) Gluc o s e t oler ance t est a r ea unde r t he c u r v e (A U C) c al cula ted f r o m indi vidual gluc ose t oler ance t es ts of mic e f ed a ND or HFD . D at a a r e p r esen t ed as me an ±S D , n=5. (C ) W eigh t in gr ams of mice f ed a ND or HFD D a t a are pr esen ted as mean ± SD , n=5. Sup plemen t ar y Fig 5: Alt er na t iv e HuR KD (Y A KD ) c on st ruct e nrichm en t analysis sugg es ts o ff t ar get eff ect s (A ) Gene set enrichmen t analy s i s u s in g eVI T T A t oolbo x af t er Y A KD of DE G s a t LogF C 1 an d -log10 adjus t ed p < 0 .05. Enr ic hed t erms include splici n g and RNA met abolis m as w ell as DNA dama g e r epair , cel l c heckpoin t ac t i v a tion and r edu c ed cell adhen s ion and pr olif er a ti on. ( B ) Sca t t er plot s howing DS G s a r ound AS in Y A KD e v e n t s i n 5 p a t t e r n s s h o w i n g almos t no di f f e r ential ly s p l iced g ene s a t FD R < 0.05 and 0.2 < ΔZiKU < -0.2. .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 22, 2025. ; https://doi.org/10.1101/2025.08.08.668956doi: bioRxiv preprint Sup plemen t ar y T able 1: Ov er r epr es en t ation analy s is of DSG s in pr i mary os t e o c yt es cultu r ed in HG con ditions. Sup plemen t ar y T able 2: T op 20 R NA binding mot i f s ar o und diff eren tiall y s plic ed t r ans c r ipts i n p rimary os t eocyt es cultu r ed in HG con ditions. Sup plemen t ar y T able 3: Ov er r epr es en t ation analy s is of DE G S in aft er HuR KD . Sup plemen t ar y T able 4: D S Gs, ov e r r epr esen t a tion anal y sis of DSGs and GOC C enr ic h men t analy s is aft er H uR KD . Sup plemen t ar y T able 5: P ri m e rs s e q ue n c e s, s h RN A co nst r uc t de s ig n s a nd antibodies used for Western blotting. Sup plemen t ar y T able 6: r MA TS outpu t fr om p rima ry os t eocyt es c ult ur ed in HG c ondit i o ns . Sup plemen t ar y T able 7: r MAP S out put fr om p rima ry ost eoc yt es cultur ed i n H G c ondit i o ns . Sup plemen t ar y T able 8: r MA TS outpu t aft er HuR KD . .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 22, 2025. ; https://doi.org/10.1101/2025.08.08.668956doi: bioRxiv preprint .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 22, 2025. ; https://doi.org/10.1101/2025.08.08.668956doi: bioRxiv preprint .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 22, 2025. ; https://doi.org/10.1101/2025.08.08.668956doi: bioRxiv preprint .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 22, 2025. ; https://doi.org/10.1101/2025.08.08.668956doi: bioRxiv preprint .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 22, 2025. ; https://doi.org/10.1101/2025.08.08.668956doi: bioRxiv preprint .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 22, 2025. ; https://doi.org/10.1101/2025.08.08.668956doi: bioRxiv preprint .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 22, 2025. ; https://doi.org/10.1101/2025.08.08.668956doi: bioRxiv preprint .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 22, 2025. ; https://doi.org/10.1101/2025.08.08.668956doi: bioRxiv preprint .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 22, 2025. ; https://doi.org/10.1101/2025.08.08.668956doi: bioRxiv preprint .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 22, 2025. ; https://doi.org/10.1101/2025.08.08.668956doi: bioRxiv preprint .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 22, 2025. ; https://doi.org/10.1101/2025.08.08.668956doi: bioRxiv preprint .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 22, 2025. ; https://doi.org/10.1101/2025.08.08.668956doi: bioRxiv preprint .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 22, 2025. ; https://doi.org/10.1101/2025.08.08.668956doi: bioRxiv preprint .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 22, 2025. ; https://doi.org/10.1101/2025.08.08.668956doi: bioRxiv preprint