Histone H3K9 Lactylation Confers Temozolomide Resistance in Glioblastoma via LUC7L2-Mediated MLH1 Intron Retention.

Temozolomide (TMZ) resistance remains the major obstacle in the treatment of glioblastoma (GBM). Lactylation is a novel post-translational modification that is involved in various tumors. However, whether lactylation plays a role in GBM TMZ resistance remains unclear. Here it is found that histone H3K9 lactylation (H3K9la) confers TMZ resistance in GBM via LUC7L2-mediated intron 7 retention of MLH1. Mechanistically, lactylation is upregulated in recurrent GBM tissues and TMZ-resistant cells, and is mainly concentrated in histone H3K9. Combined multi-omics analysis, including CUT&Tag, SLAM-seq, and RNA-seq, reveals that H3K9 lactylation is significantly enriched in the LUC7L2 promoter and activates LUC7L2 transcription to promote its expression. LUC7L2 mediates intron 7 retention of MLH1 to reduce MLH1 expression, and thereby inhibit mismatch repair (MMR), ultimately leading to GBM TMZ resistance. Of note, it is identified that a clinical anti-epileptic drug, stiripentol, which can cross the blood-brain barrier and inhibit lactate dehydrogenase A/B (LDHA/B) activity, acts as a lactylation inhibitor and renders GBM cells more sensitive to TMZ in vitro and in vivo. These findings not only shed light on the mechanism of lactylation in GBM TMZ resistance but also provide a potential combined therapeutic strategy for clinical GBM treatment.

IRP1 mediated ferroptosis reverses temozolomide resistance in glioblastoma via affecting LCN2/FPN1 signaling axis depended on NFKB2.

The prognosis of glioblastoma (GBM) patients is poor, and temozolomide (TMZ) resistance has become an important obstacle to its treatment effect. A growing number of researches have revealed the special characteristics of iron metabolism in GBM chemosensitivity. Iron regulatory protein 1 (IRP1) is an important protein for maintaining intracellular iron homeostasis. IRP1 has been indicated to have additional vital roles beyond its conventional metabolic activity, but the underlying mechanisms and biological consequences remain elusive. Here, we unprecedentedly demonstrated that amplifying IRP1 signals can reverse TMZ resistance and suppress tumor growth in vivo via inhibiting NFKB2 in the noncanonical NF-κB signaling pathway. In addition, we identified that NFKB2 affected TMZ sensitivity of GBM by modulating the expression of LCN2 and FPN1. Taken together, this study established a role for the IRP1/NFKB2 pathway in regulating LCN2/FPN1 signaling axis among the progression of TMZ resistance, suggesting a potential innovative GBM therapeutic strategy.

Polyhedral Oligomeric Silsesquioxane-Based Nanoparticles for Efficient Chemotherapy of Glioblastoma.

Glioblastoma (GBM) is the most common lethal brain tumor with dismal treatment outcomes and poor response to chemotherapy. As the regulatory center of cytogenetics and metabolism, most tumor chemotherapeutic molecules exert therapeutic effects in the nucleus. Nanodrugs showing the nuclear aggregation effect are expected to eliminate and fundamentally suppress tumor cells. In this study, a nanodrug delivery system based on polyhedral oligomeric silsesquioxane (POSS) is introduced to deliver drugs into the nuclei of GBM cells, effectively enhancing the therapeutic efficacy of chemotherapy. The nanoparticles are modified with folic acid and iRGD peptides molecules to improve their tumor cell targeting and uptake via receptor-mediated endocytosis. Nuclear aggregation allows for the direct delivery of chemotherapeutic drug temozolomide (TMZ) to the tumor cell nuclei, resulting in more significant DNA damage and inhibition of tumor cell proliferation. Herein, TMZ-loaded POSS nanoparticles can significantly improve the survival of GBM-bearing mice. Therefore, the modified POSS nanoparticles may serve as a promising drug-loaded delivery platform to improve chemotherapy outcomes in GBM patients.

Nonconforming gender expression and insufficient sleep among adolescents during COVID-19 school closure and after school reopening.

BACKGROUND: Gender nonconformity (GNC) (i.e., gender expression that differs from gender role expectations for feminine or masculine appearance and behavior) is an under-researched area of adolescent sleep health. The COVID-19 lockdown offers an opportunity to understand how the effect of GNC on adolescent health outcomes changes between school closure and reopening. METHODS: We conducted a cross-sectional study in Shanghai, China, in 2020. The sample size for analysis was 3,265. The age-specific insufficient sleep was estimated according to National Sleep Foundation's sleep duration recommendations. The self-perceived and self-rated GNC were measured by the two items "On the same scale that goes from 100% as a girl to 100% as a boy, where do you think others see you?" and "On a scale that goes from feeling 100% like a girl to feeling 100% like a boy, where do you see yourself?", and birth sex. In addition, we calculated sex-stratified adjusted odds ratios (AORs) of insufficient sleep for students with high and moderate GNC compared to students with low GNC. Finally, we measured the AORs with self-perceived and self-rated GNC during COVID-19 school closure and reopening. RESULTS: Among 3,265 students in grade 6-12 in the analytic sample, 1,567(48.0%) were assigned female at birth (AFAB), 3,188 (97.6%) Han, and 1,921(58.8%) in grade 6-9. Among AFAB students, high self-perceived GNC was significantly associated with insufficient sleep (AOR,1.65; 95%CI,1.30-2.09) during school closure. Insufficient sleep was associated with high self-rated GNC (AOR,1.73; 95%CI,1.23-2.44) and moderate self-rated GNC (AOR,1.69; 95%CI,1.29-2.22) during school closure. After school reopening, neither self-perceived nor self-rated GNC was associated with insufficient sleep among AFAB students. Among assigned male at birth (AMAB) students, none of the two kinds of GNC was associated with insufficient sleep in the two periods during the COVID-19 pandemic. CONCLUSIONS: This study suggests GNC is only associated with insufficient sleep among AFAB students during school closure. Furthermore, the association is nonsignificant among AMAB students. These findings indicate that GNC-related stigma within the family could be a risk factor for insufficient sleep among AFAB adolescents.

Comparing the Perceptions of Gender Norms among Adolescents with Different Sibling Contexts in Shanghai, China.

Individuals' gender development is influenced by the characteristics of personal and contextual environments. However, the role of sibling contexts in shaping gender norms has rarely been studied among Chinese youth at early adolescence as most of them were the only child. The aim of this paper is to compare perceived gender norms among adolescents aged 10-14 with different sibling configurations, to help inform and tailor guidance for sexual and reproductive health education in the future. We used the Global Early Adolescent Study baseline data collected from Shanghai, China. The sample for analysis was 1615 students. We used univariate analysis and multivariate ordinal logistic regression to compare perceived gender-stereotyped traits and gender role attitudes, stratified by age and sex. The results showed that sibling context was more influential for boys than girls at early adolescence in their gender socialization process. Among boys those who were with mixed-sex siblings scored higher on gender-stereotyped traits (ORonly-childvs. mixed-sex siblings = 0.67, 95% CI: 0.48-0.94, p = 0.019; ORsame-sex siblingsvs. mixed-sex siblings = 0.59, 95% CI: 0.37-0.96, p = 0.033). Younger early adolescents aged 10-12 who were the only child or who had mixed-sex siblings perceived more traditional gender role attitudes than those living with same-sex siblings (ORonly-childvs. same-sex siblings = 1.71, 95% CI: 1.06-2.75, p = 0.028; ORmixed-sex siblingsvs. same-sex siblings = 1.74, 95% CI: 1.03-2.94, p = 0.037). Comprehensive sexuality education with gender and power components being well addressed, both in and out of the family, is needed to provide extra gender-inclusive and gender-egalitarian environments for youth.

Relationship between gender role attitudes and depression among adolescents / 中国学校卫生

Objective@#To explore the relationship between gender role attitudes (GRA) and adolescent depression to provide evidence for the promotion of mental health among adolescents.@*Methods@#A total of 1 549 students from grades 6 to 8 in three public middle schools in Jing an District of Shanghai were selected by stratified cluster sampling. The baseline and follow up surveys were conducted by anonymous electronic questionnaire on mobile tablets from November to December in 2017 and 2018, respectively. The GRA Scale and the Depression Scale were used to collect subjects attitudes towards traditional role assignment and power inequality between men and woman and depression in family and sexual relationships. The multivariate Generalized Estimating Equations (GEE) model was adopted to examine the relationship between GRA and depression.@*Results@#The mean score of the GRA Scale was (2.52±0.83) and (2.29±0.86), while the score of Depression Scale was (15.92±5.08) and (16.48±5.29), in the baseline and follow up survey, respectively ( P <0.05). After controlling the covariates of age, bullied experience, body image, social cohesion, etc., the multivariate GEE model indicated traditional GRA was significantly associated with a higher risk of depression among both boys and girls ( β boys =0.38, β girls =0.41, P <0.05).@*Conclusion@#Traditional GRA may increase the risk of depression in adolescents, suggesting that cultivating a positive and equal GRA among this population group may help to prevent depression.

Transformation of the sp2 Carbanion to Carbene with Subsequent 1,1-Migratory Insertion and Nucleophilic Substitution in Rare-Earth Metal Chemistry.

The development of Fischer-type electrophilic carbene chemistry with early transition metals has been a great challenge due to the fact that such metals in their high oxidation states lack the d electrons to stabilize the electrophilic carbene. Herein, we disclose the first experimental and theoretical findings of in situ transformation of an sp2 carbanion to a Fischer-type electrophilic carbene with rare-earth metals in their high oxidation state with a d0 electron via electron transfer. The carbene may undergo 1,1-migratory insertion into an adjacent RE-C(sp3) bond, and an unprecedented ring opening of the indole ring of the ligand occurs when the carbenes undergo nucleophilic substitution with a special organolithium reagent o-Me2NC6H4CH2Li. The key to success is the uniquely tailored novel ligand systems featuring a suitable conjugate building block (-C═C-C═N) bearing an sp2 carbanion connected to the rare-earth metal center.

The effect of COVID-19 school closures on adolescent sleep duration: an uncontrolled before-after study.

BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic affected almost 1.6 billion students or more than 90% of learners globally. However, the effect of school closures during COVID-19 pandemic on adolescent sleep duration remains unclear. METHODS: We undertook a cross-sectional electronic survey in six junior and senior high schools in Shanghai, China from late June to early July 2020. We evaluated the changes of sleep duration on weekdays by comparing sleep duration hours and insufficient sleep (< 9 h for children aged 6-13 years or < 8 h for teenagers aged 14-17 years) in COVID-19 school closures and after school reopening. We also investigated possible sex differences in the changes of sleep duration. RESULTS: A total of 3265 students completed the survey, the mean age was 14.56 ± 1.99 years, 1567 (47.99%) were girls and 1344 (41.17%) were in grades 10-12. The overall sleep duration decreased from 8.88 h in school closures to 7.77 h after school reopening, and the change (difference: - 1.11 h; 95%CI: - 1.16, - 1.07; P < 0.001) was statistically significant. The prevalence of insufficient sleep increased sharply from 21.10 to 63.98%, and the change (ratio:3.03; 95%CI:2.84, 3.23; P < 0.001) was statistically significant. Besides, the changes were greater in girls than in boys. CONCLUSION: Results of this study revealed that sleep duration was longer and percentage of sufficient sleep was higher during COVID-19 school closures in adolescent students.

Pentraxin 3 secreted by human adipose-derived stem cells promotes dopaminergic neuron repair in Parkinson's disease via the inhibition of apoptosis.

Although adipose-derived human mesenchymal stem cell (hADSC) transplantation has recently emerged as a promising therapeutic modality for Parkinson's disease (PD), its underlying mechanism of action has not been fully elucidated. This study evaluated the therapeutic effects of stereotaxic injection of hADSCs in the striatum of the 6-OHDA-induced mouse model. Furthermore, an in vitro PD model was constructed using tissue-organized brain slices. The therapeutic effect was also evaluated using a co-culture of the hADSCs and 6-OHDA-treated brain slice. The analysis of hADSC exocrine proteins using RNA-sequencing, human protein cytokine arrays, and label-free quantitative proteomics identified key extracellular factors in the hADSC secretion environment. The degeneration and apoptosis of the dopaminergic neurons were measured in the PD samples in vivo and in vitro, and the beneficial effects were evaluated using quantitative reverse transcription-polymerase chain reaction, western blotting, Fluoro-Jade C, TUNEL assay, and immunofluorescence analysis. This study found that hADSCs protected the dopaminergic neurons in the in vivo and vitro models. We identified Pentraxin 3 (PTX3) as a key extracellular factor in the hADSC secretion environment. Moreover, we found that human recombinant PTX3 (rhPTX3) treatment could rescue the pathophysiological behavior of the PD mice in vivo, prevent dopaminergic neuronal death, and increase neuronal terminals in the ventral tegmental area + substantia nigra pars compacta and striatum in the PD brain slices in vitro. Furthermore, testing of the pro-apoptotic markers in the PD mouse brain following rhPTX3 treatment revealed that rhPTX3 can prevent apoptosis and degeneration of the dopaminergic neurons. This study discovered that PTX3, a hADSC-secreted protein, potentially protected the dopaminergic neurons against apoptosis and degeneration during PD progression and improved motor performance in PD mice, indicating the possible mechanism of action of hADSC replacement therapy for PD. Thus, our study discovered potential translational implications for the development of PTX3-based therapeutics for PD.

Influence of impulsivity on adolescents pornography use / 中国学校卫生

Objective@#To explore the influence of impulsive traits of adolescent on pornography use through the analysis of the Global Early Adolescent Study(GEAS) longitudinal survey data collected in Shanghai, and to provide evidence for adolescent sexual health promotion.@*Methods@#Totally 1 512 students from grades 6 to 8 in three middle schools in a district of Shanghai were selected for the present study by stratified cluster sampling method. Baseline and two-wave follow-up investigation were conducted between 2017 and 2020. Three waves of pornography use and social demographic information were collected using electronic questionnaire through tablets while impulsivity were tested only once during the follow-up in 2018 using the paper and pencil based Barratt Impulsivity Scale, version11 (BIS-11). General statistical description and panel data statistical description, as well as multilevel mixed effect linear model were used to do the analysis.@*Results@#About 32.7% (494) of the adolescents reported the experience of watching pornography in threewave analysis. Boys were more impulsive than girls in the motor subscale (16.33±3.25, 15.66±2.93, t=4.13, P<0.01) while girls were more impulsive than boys in the nonplanning subscale (23.65±5.11, 22.83±5.21, t=-3.03, P<0.01). Mixed effect linear model result showed that impulsivity was correlated with pornography use(β=0.001, P<0.01); higher impulsivity in motor and attention were correlated with more frequent pornography use(P<0.01). Being female, perceived more parental awareness and perceived care from school adults would decrease the use of pornography while spent more than 3 hours on internet would increase the use of pornography (P<0.01).@*Conclusion@#There are stable correlations between impulsivity and pornography use. It is urgent to equip young adolescents with the necessary ability to distinguish the good from the bad in the mass and internet media world.

Association between adverse childhood experiences and adolescent alcohol use / 中国学校卫生

Objective@#To explore the association between adverse childhood experiences (ACEs) and adolescent alcohol use and to provide evidence for prevention and intervention.@*Methods@#A total of 1 550 students from grades 6 to 8 in three public middle schools in a district of Shanghai were selected by stratified cluster sampling method. The baseline and follow up investigation were conducted by anonymous electronic questionnaire in mobile tablet from November to December in 2017 and 2018, respectively. The latent class analysis (LCA) was applied for the classification of ACEs, while the multivariate generalized estimating Equations (GEE) model was adopted to examine the association between ACEs and adolescent alcohol use.@*Results@#The ACEs was divided into 3 classes by LCA: high exposure (8.97%), abuse and neglect (38.97%), low exposure (52.06%). The multivariate GEE model indicates the risk of alcohol drinking among adolescents in high exposure and abuse and neglect classes were significantly higher than their counterparts in low exposure class ( OR=2.65, 95%CI=1.72-4.07; OR=1.50, 95%CI =1.14-1.96, respectively).@*Conclusion@#The effect of ACEs on alcohol use may vary across different latent classes. Supportive childhood environment may contribute to decrease the risk of adolescent drinking behavior.

Heterosexual romantic expectation of early adolescents and its influencing factors / 中国学校卫生

Objective@#To explore the heterosexual romantic expectation in early adolescence(10-14 years)and its influencing factors, and to provide a reference for instructing children of early puberty to establish a positive interpersonal relationship(including heterosexual relationship) and improving their healthy development.@*Methods@#Stratified cluster sampling method was adopted to recruit students of grade 6-8 in three middle schools of Shanghai during November to December, 2017. Students were surveyed anonymously using Computer Assisted Self-Interview approach via the tablets. The collected data included the information on demographic, families, peers, use of the media, community cohesion and heterosexual romantic expectation. The ttest, Chi-square test, ANOVA and multiple linear regression were used to explore the relationships between potential factors and heterosexual romantic expectation.@*Results@#The mean score of heterosexual romantic expectation was 2.55. Score of the boys was higher than that of girls (2.62 vs. 2.47, t=2.65, P<0.05). There was no statistically significant difference between the scores of the ages before 12 years old (10-11: 2.34, 12: 2.28), but a significant increase of the scores along the age after 12 years old(13: 2.69, 14: 3.05). Multiple linear regression suggested that respondents with older age, poorer caregiver-child relationship, higher proportion of friends of the opposite sex, more friends that thought having boyfriends/girlfriends was important, more TV/movie use and lower community cohesion had higher score of heterosexual romantic expectation of early adolescents(P<0.05).@*Conclusion@#The early adolescents had relative conservative views on the heterosexual romantic relationships of adolescents of their own age while the boys were more permissive than girls. The heterosexual romantic expectation improved significantly after 12 years and may be influenced by factors of family, peers, media and community.

Relationship between different roles in campus bullying and depression / 中国学校卫生

Objective@#To explore the relationship between different roles in campus bullying and depression among adolescents.@*Methods@#Stratified cluster sampling method was used to select adolescents in grade 6-8 from three middle schools of Shanghai during November and December of 2017. Adolescents were surveyed anonymously using computer assisted self-interview approach via the tablets. Information including demographic characteristics, depression, and bullying was collected. The chi-square test and multivariate Logistic regressions were performed to explore the relationship between bullying roles and depression.@*Results@#About 75.74% of the respondents were involved in bullying, including 456(27.65%) bystanders, 559(33.90%) victims, 33(2.00%) perpetrators, and 201 (12.19%) perpetrator-victims. There were 1 022(61.98%) respondents in the low depression group and 627 (38.02%) in the high depression group. Multivariate logistic regression showed that the four sub-groups involved in bullying all had higher level of depression than those uninvolved, with the perpetrator-victims (OR=4.77, 95%CI=3.27-6.96) and the victims (OR=3.66, 95%CI=2.71-4.94) had more depressive symptoms.@*Conclusion@#Different roles in campus bullying associates with more depressive symptoms, including perpetrators, victims, perpetrator-victims, and the bystanders.

Bcl3 Phosphorylation by Akt, Erk2, and IKK Is Required for Its Transcriptional Activity.

Unlike prototypical IκB proteins, which are inhibitors of NF-κB RelA, cRel, and RelB dimers, the atypical IκB protein Bcl3 is primarily a transcriptional coregulator of p52 and p50 homodimers. Bcl3 exists as phospho-protein in many cancer cells. Unphosphorylated Bcl3 acts as a classical IκB-like inhibitor and removes p50 and p52 from bound DNA. Neither the phosphorylation site(s) nor the kinase(s) phosphorylating Bcl3 is known. Here we show that Akt, Erk2, and IKK1/2 phosphorylate Bcl3. Phosphorylation of Ser33 by Akt induces switching of K48 ubiquitination to K63 ubiquitination and thus promotes nuclear localization and stabilization of Bcl3. Phosphorylation by Erk2 and IKK1/2 of Ser114 and Ser446 converts Bcl3 into a transcriptional coregulator by facilitating its recruitment to DNA. Cells expressing the S114A/S446A mutant have cellular proliferation and migration defects. This work links Akt and MAPK pathways to NF-κB through Bcl3 and provides mechanistic insight into how Bcl3 functions as an oncoprotein through collaboration with IKK1/2, Akt, and Erk2.

Prevalence and contributing factors to attention deficit hyperactivity disorder: a study of five- to fifteen-year-old children in Zhabei District, Shanghai.

INTRODUCTION: This work aims to understand the features among 5- to 15-year-old children with attention deficit hyperactivity disorder (ADHD) in Zhabei District in Shanghai. METHODS: Children with ADHD were studied using general background questionnaire, ADHD symptom rating questionnaire, and cluster-stratified sampling. A total of 9,900 valid questionnaires were utilized in this study. We conducted diagnostic interviews with suspected ADHD children and their parents using the criteria of the Diagnostic and Statistical Manual of Mental Disorders (4th Edition) for ADHD. RESULTS: The prevalence rate of ADHD among the children was 4.6%, of which 2.4%, 0.4%, and 1.8% had ADHD-I ADHD-HI, and ADHD-C types, respectively. The prevalence rates in boys and girls were 6.6% and 2.7% (ratio, 2.41 : 1), respectively. Significant differences in prevalence rate were found among children with different age groups and ADHD types. Children aged 7-10 years had the highest prevalence rate (6.3%). Externally, residence children had higher prevalence than local residents. Significant differences in prevalence rate were also found among children with parents having different educational and socioeconomic level. DISCUSSION: The prevalence of ADHD-HI was higher than the other two types. The highest prevalence was observed in 7- to 10-year-old children. The influential factors of ADHD prevalence were age, gender, and educational level.

A structural basis for IκB kinase 2 activation via oligomerization-dependent trans auto-phosphorylation.

Activation of the IκB kinase (IKK) is central to NF-κB signaling. However, the precise activation mechanism by which catalytic IKK subunits gain the ability to induce NF-κB transcriptional activity is not well understood. Here we report a 4 Å x-ray crystal structure of human IKK2 (hIKK2) in its catalytically active conformation. The hIKK2 domain architecture closely resembles that of Xenopus IKK2 (xIKK2). However, whereas inactivated xIKK2 displays a closed dimeric structure, hIKK2 dimers adopt open conformations that permit higher order oligomerization within the crystal. Reversible oligomerization of hIKK2 dimers is observed in solution. Mutagenesis confirms that two of the surfaces that mediate oligomerization within the crystal are also critical for the process of hIKK2 activation in cells. We propose that IKK2 dimers transiently associate with one another through these interaction surfaces to promote trans auto-phosphorylation as part of their mechanism of activation. This structure-based model supports recently published structural data that implicate strand exchange as part of a mechanism for IKK2 activation via trans auto-phosphorylation. Moreover, oligomerization through the interfaces identified in this study and subsequent trans auto-phosphorylation account for the rapid amplification of IKK2 phosphorylation observed even in the absence of any upstream kinase.

Interaction between the RNA binding domains of Ser-Arg splicing factor 1 and U1-70K snRNP protein determines early spliceosome assembly.

It has been widely accepted that the early spliceosome assembly begins with U1 small nuclear ribonucleoprotein (U1 snRNP) binding to the 5' splice site (5'SS), which is assisted by the Ser/Arg (SR)-rich proteins in mammalian cells. In this process, the RS domain of SR proteins is thought to directly interact with the RS motif of U1-70K, which is subject to regulation by RS domain phosphorylation. Here we report that the early spliceosome assembly event is mediated by the RNA recognition domains (RRM) of serine/arginine-rich splicing factor 1 (SRSF1), which bridges the RRM of U1-70K to pre-mRNA by using the surface opposite to the RNA binding site. Specific mutation in the RRM of SRSF1 that disrupted the RRM-RRM interaction also inhibits the formation of spliceosomal E complex and splicing. We further demonstrate that the hypo-phosphorylated RS domain of SRSF1 interacts with its own RRM, thus competing with U1-70K binding, whereas the hyper-phosphorylated RS domain permits the formation of a ternary complex containing ESE, an SR protein, and U1 snRNP. Therefore, phosphorylation of the RS domain in SRSF1 appears to induce a key molecular switch from intra- to intermolecular interactions, suggesting a plausible mechanism for the documented requirement for the phosphorylation/dephosphorylation cycle during pre-mRNA splicing.

SR proteins in vertical integration of gene expression from transcription to RNA processing to translation.

SR proteins have been studied extensively as a family of RNA-binding proteins that participate in both constitutive and regulated pre-mRNA splicing in mammalian cells. However, SR proteins were first discovered as factors that interact with transcriptionally active chromatin. Recent studies have now uncovered properties that connect these once apparently disparate functions, showing that a subset of SR proteins seem to bind directly to the histone 3 tail, play an active role in transcriptional elongation, and colocalize with genes that are engaged in specific intra- and interchromosome interactions for coordinated regulation of gene expression in the nucleus. These transcription-related activities are also coupled with a further expansion of putative functions of specific SR protein family members in RNA metabolism downstream of mRNA splicing, from RNA export to stability control to translation. These findings, therefore, highlight the broader roles of SR proteins in vertical integration of gene expression and provide mechanistic insights into their contributions to genome stability and proper cell-cycle progression in higher eukaryotic cells.

Regulation of SR protein phosphorylation and alternative splicing by modulating kinetic interactions of SRPK1 with molecular chaperones.

Phosphorylation is essential for the SR family of splicing factors/regulators to function in constitutive and regulated pre-mRNA splicing; yet both hypo- and hyperphosphorylation of SR proteins are known to inhibit splicing, indicating that SR protein phosphorylation must be tightly regulated in the cell. However, little is known how SR protein phosphorylation might be regulated during development or in response to specific signaling events. Here, we report that SRPK1, a ubiquitously expressed SR protein-specific kinase, directly binds to the cochaperones Hsp40/DNAjc8 and Aha1, which mediate dynamic interactions of the kinase with the major molecular chaperones Hsp70 and Hsp90 in mammalian cells. Inhibition of the Hsp90 ATPase activity induces dissociation of SRPK1 from the chaperone complexes, which can also be triggered by a stress signal (osmotic shock), resulting in translocation of the kinase from the cytoplasm to the nucleus, differential phosphorylation of SR proteins, and alteration of splice site selection. These findings connect the SRPK to the molecular chaperone system that has been implicated in numerous signal transduction pathways and provide mechanistic insights into complex regulation of SR protein phosphorylation and alternative splicing in response to developmental cues and cellular signaling.

Regulated cellular partitioning of SR protein-specific kinases in mammalian cells.

Reversible phosphorylation of the SR family of splicing factors plays an important role in pre-mRNA processing in the nucleus. Interestingly, the SRPK family of kinases specific for SR proteins is localized in the cytoplasm, which is critical for nuclear import of SR proteins in a phosphorylation-dependent manner. Here, we report molecular dissection of the mechanism involved in partitioning SRPKs in the cytoplasm. Common among all SRPKs, the bipartite kinase catalytic core is separated by a unique spacer sequence. The spacers in mammalian SRPK1 and SRPK2 share little sequence homology, but they function interchangeably in restricting the kinases in the cytoplasm. Removal of the spacer in SRPK1 had little effect on the kinase activity, but it caused a quantitative translocation of the kinase to the nucleus and consequently induced aggregation of splicing factors in the nucleus. Rather than carrying a nuclear export signal as suggested previously, we found multiple redundant signals in the spacer that act together to anchor the kinase in the cytoplasm. Interestingly, a cell cycle signal induced nuclear translocation of the kinase at the G2/M boundary. These findings suggest that SRPKs may play an important role in linking signaling to RNA metabolism in higher eukaryotic cells.