SAM68 is a physiological regulator of SMN2 splicing in spinal muscular atrophy

Spinal muscular atrophy (SMA) is a neurodegenerative disease caused by loss of motor neurons in patients with null mutations in the SMN1 gene. The almost identical SMN2 gene is unable to compensate for this deficiency because of the skipping of exon 7 during pre-messenger RNA (mRNA) processing. Although several splicing factors can modulate SMN2 splicing in vitro, the physiological regulators of this disease-causing event are unknown. We found that knockout of the splicing factor SAM68 partially rescued body weight and viability of SMAΔ7 mice. Ablation of SAM68 function promoted SMN2 splicing and expression in SMAΔ7 mice, correlating with amelioration of SMA-related defects in motor neurons and skeletal muscles. Mechanistically, SAM68 binds to SMN2 pre-mRNA, favoring recruitment of the splicing repressor hnRNP A1 and interfering with that of U2AF65 at the 3' splice site of exon 7. These findings identify SAM68 as the first physiological regulator of SMN2 splicing in an SMA mouse model

The Caenorhabditis elegans Gene mfap-1 Encodes a Nuclear Protein That Affects Alternative Splicing

RNA splicing is a major regulatory mechanism for controlling eukaryotic gene expression. By generating various splice isoforms from a single pre–mRNA, alternative splicing plays a key role in promoting the evolving complexity of metazoans. Numerous splicing factors have been identified. However, the in vivo functions of many splicing factors remain to be understood. In vivo studies are essential for understanding the molecular mechanisms of RNA splicing and the biology of numerous RNA splicing-related diseases. We previously isolated a Caenorhabditis elegans mutant defective in an essential gene from a genetic screen for suppressors of the rubberband Unc phenotype of unc-93(e1500) animals. This mutant contains missense mutations in two adjacent codons of the C. elegans microfibrillar-associated protein 1 gene mfap-1. mfap-1(n4564 n5214) suppresses the Unc phenotypes of different rubberband Unc mutants in a pattern similar to that of mutations in the splicing factor genes uaf-1 (the C. elegans U2AF large subunit gene) and sfa-1 (the C. elegans SF1/BBP gene). We used the endogenous gene tos-1 as a reporter for splicing and detected increased intron 1 retention and exon 3 skipping of tos-1 transcripts in mfap-1(n4564 n5214) animals. Using a yeast two-hybrid screen, we isolated splicing factors as potential MFAP-1 interactors. Our studies indicate that C. elegans mfap-1 encodes a splicing factor that can affect alternative splicing.National Natural Science Foundation (China) (Grant 30971639)United States. National Institutes of Health (Grant GM24663

A serine/arginine-rich nuclear matrix cyclophilin interacts with the C-terminal domain of RNA polymerase II

The largest subunit of RNA polymerase II shows a striking difference in the degree of phosphorylation, depending on its functional state: initiating and elongating polymerases are unphosphorylated and highly phosphorylated respectively. Phosphorylation mostly occurs at the C-terminal domain (CTD), which consists of a repetitive heptapeptide structure. Using the yeast two-hybrid system, we have selected for mammalian proteins that interact with the phosphorylated CTD of mammalian RNA polymerase II. A prominent isolate, designated SRcyp/CASP10, specifically interacts with the CTD not only in vivo but also in vitro. It contains a serine/arginine-rich (SR) domain, similar to that found in the SR protein family of pre-mRNA splicing factors, which is required for interaction with the CTD. Most remarkably, the N-terminal region of SRcyp includes a peptidyl-prolyl cis-trans isomerase domain characteristic of immunophilins/cyclophilins (Cyp), a protein family implicated in protein folding, assembly and transport. SRcyp is a nuclear protein with a characteristic distribution in large irregularly shaped nuclear speckles and co-localizes perfectly with the SR domaincontaining splicing factor SC35. Recent independent investigations have provided complementary data, such as an association of the phosphorylated form of RNA polymerase II with the nuclear speckles, impaired splicing in a CTD deletion background and inhibition of in vitro splicing by CTD peptides. Taken together, these data indicate that factors directly or indirectly involved in splicing are associated with the elongating RNA polymerases, from where they might translocate to the nascent transcripts to ensure efficient splicing, concomitant with transcriptio

De novo prediction of PTBP1 binding and splicing targets reveals unexpected features of its RNA recognition and function.

The splicing regulator Polypyrimidine Tract Binding Protein (PTBP1) has four RNA binding domains that each binds a short pyrimidine element, allowing recognition of diverse pyrimidine-rich sequences. This variation makes it difficult to evaluate PTBP1 binding to particular sites based on sequence alone and thus to identify target RNAs. Conversely, transcriptome-wide binding assays such as CLIP identify many in vivo targets, but do not provide a quantitative assessment of binding and are informative only for the cells where the analysis is performed. A general method of predicting PTBP1 binding and possible targets in any cell type is needed. We developed computational models that predict the binding and splicing targets of PTBP1. A Hidden Markov Model (HMM), trained on CLIP-seq data, was used to score probable PTBP1 binding sites. Scores from this model are highly correlated (ρ = -0.9) with experimentally determined dissociation constants. Notably, we find that the protein is not strictly pyrimidine specific, as interspersed Guanosine residues are well tolerated within PTBP1 binding sites. This model identifies many previously unrecognized PTBP1 binding sites, and can score PTBP1 binding across the transcriptome in the absence of CLIP data. Using this model to examine the placement of PTBP1 binding sites in controlling splicing, we trained a multinomial logistic model on sets of PTBP1 regulated and unregulated exons. Applying this model to rank exons across the mouse transcriptome identifies known PTBP1 targets and many new exons that were confirmed as PTBP1-repressed by RT-PCR and RNA-seq after PTBP1 depletion. We find that PTBP1 dependent exons are diverse in structure and do not all fit previous descriptions of the placement of PTBP1 binding sites. Our study uncovers new features of RNA recognition and splicing regulation by PTBP1. This approach can be applied to other multi-RRM domain proteins to assess binding site degeneracy and multifactorial splicing regulation

A splicing-dependent transcriptional checkpoint associated with prespliceosome formation

There is good evidence for functional interactions between splicing and transcription in eukaryotes, but how and why these processes are coupled remain unknown. Prp5 protein (Prp5p) is an RNA-stimulated adenosine triphosphatase (ATPase) required for prespliceosome formation in yeast. We demonstrate through in vivo RNA labeling that, in addition to a splicing defect, the prp5-1 mutation causes a defect in the transcription of intron-containing genes. We present chromatin immunoprecipitation evidence for a transcriptional elongation defect in which RNA polymerase that is phosphorylated at Ser5 of the largest subunit’s heptad repeat accumulates over introns and that this defect requires Cus2 protein. A similar accumulation of polymerase was observed when prespliceosome formation was blocked by a mutation in U2 snRNA. These results indicate the existence of a transcriptional elongation checkpoint that is associated with prespliceosome formation during cotranscriptional spliceosome assembly. We propose a role for Cus2p as a potential checkpoint factor in transcription

A Role for Pre-mRNA-PROCESSING PROTEIN 40C in the Control of Growth, Development, and Stress Tolerance in Arabidopsis thaliana

Because of their sessile nature, plants have adopted varied strategies for growing and reproducing in an ever-changing environment. Control of mRNA levels and pre-mRNA alternative splicing are key regulatory layers that contribute to adjust and synchronize plant growth and development with environmental changes. Transcription and alternative splicing are thought to be tightly linked and coordinated, at least in part, through a network of transcriptional and splicing regulatory factors that interact with the carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II. One of the proteins that has been shown to play such a role in yeast and mammals is pre-mRNA-PROCESSING PROTEIN 40 (PRP40, also known as CA150, or TCERG1). In plants, members of the PRP40 family have been identified and shown to interact with the CTD of RNA Pol II, but their biological functions remain unknown. Here, we studied the role of AtPRP40C, in Arabidopsis thaliana growth, development and stress tolerance, as well as its impact on the global regulation of gene expression programs. We found that the prp40c knockout mutants display a late-flowering phenotype under long day conditions, associated with minor alterations in red light signaling. An RNA-seq based transcriptome analysis revealed differentially expressed genes related to biotic stress responses and also differentially expressed as well as differentially spliced genes associated with abiotic stress responses. Indeed, the characterization of stress responses in prp40c mutants revealed an increased sensitivity to salt stress and an enhanced tolerance to Pseudomonas syringae pv. maculicola (Psm) infections. This constitutes the most thorough analysis of the transcriptome of a prp40 mutant in any organism, as well as the first characterization of the molecular and physiological roles of a member of the PRP40 protein family in plants. Our results suggest that PRP40C is an important factor linking the regulation of gene expression programs to the modulation of plant growth, development, and stress responses.Fil: Hernando, Carlos Esteban. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: García Hourquet, Mariano. Fundación Instituto Leloir; ArgentinaFil: de Leone, María José. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Careno, Daniel Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Iserte, Javier Alonso. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Mora Garcia, Santiago. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Yanovsky, Marcelo Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentin

Keratinocyte differentiation-dependent human papillomavirus gene regulation

Human papillomaviruses (HPVs) cause diseases ranging from benign warts to invasive cancers. HPVs infect epithelial cells and their replication cycle is tightly linked with the differentiation process of the infected keratinocyte. The normal replication cycle involves an early and a late phase. The early phase encompasses viral entry and initial genome replication, stimulation of cell division and inhibition of apoptosis in the infected cell. Late events in the HPV life cycle include viral genome amplification, virion formation, and release into the environment from the surface of the epithelium. The main proteins required at the late stage of infection for viral genome amplification include E1, E2, E4 and E5. The late proteins L1 and L2 are structural proteins that form the viral capsid. Regulation of these late events involves both cellular and viral proteins. The late viral mRNAs are expressed from a specific late promoter but final late mRNA levels in the infected cell are controlled by splicing, polyadenylation, nuclear export and RNA stability. Viral late protein expression is also controlled at the level of translation. This review will discuss current knowledge of how HPV late gene expression is regulated

Interaction of eukaryotic translation initiation factor 4G with the nuclear cap-binding complex provides a link between nuclear and cytoplasmic functions of the m7 guanosine cap

In eukaryotes the majority of mRNAs have an m7G cap that is added cotranscriptionally and that plays an important role in many aspects of mRNA metabolism. The nuclear cap-binding complex (CBC; consisting of CBP20 and CBP80) mediates the stimulatory functions of the cap in pre-mRNA splicing, 3' end formation, and U snRNA export. As little is known about how nuclear CBC mediates the effects of the cap in higher eukaryotes, we have characterized proteins that interact with CBC in HeLa cell nuclear extracts as potential mediators of its function. Using cross-linking and coimmunoprecipitation, we show that eukaryotic translation initiation factor 4G (eIF4G), in addition to its function in the cytoplasm, is a nuclear CBC-interacting protein. We demonstrate that eIF4G interacts with CBC in vitro and that, in addition to its cytoplasmic localization, there is a significant nuclear pool of eIF4G in mammalian cells in vivo. Immunoprecipitation experiments suggest that, in contrast to the cytoplasmic pool, much of the nuclear eIF4G is not associated with eIF4E (translation cap binding protein of eIF4F) but is associated with CBC. While eIF4G stably associates with spliceosomes in vitro and shows close association with spliceosomal snRNPs and splicing factors in vivo, depletion studies show that it does not participate directly in the splicing reaction. Taken together the data indicate that nuclear eIF4G may be recruited to pre-mRNAs via its interaction with CBC and accompanies the mRNA to the cytoplasm, facilitating the switching of CBC for eIF4F. This may provide a mechanism to couple nuclear and cytoplasmic functions of the mRNA cap structure

Cellular bases of the RNA metabolism dysfunction in motor neurons of a murine model of spinal muscular atrophy: Role of Cajal bodies and the nucleolus

Spinal muscular atrophy (SMA) is caused by a homozygous deletion or mutation in the survival motor neuron 1 (SMN1) gene that leads to reduced levels of SMN protein resulting in degeneration of motor neurons (MNs). The best known functions of SMN is the biogenesis of spliceosomal snRNPs. Linked to this function, Cajal bodies (CBs) are involved in the assembly of spliceosomal (snRNPs) and nucleolar (snoRNPs) ribonucleoproteins required for pre-mRNA and pre-rRNA processing. Recent studies support that the interaction between CBs and nucleoli, which are especially prominent in neurons, is essential for the nucleolar rRNA homeostasis. We use the SMN∆7 murine model of type I SMA to investigate the cellular basis of the dysfunction of RNA metabolism in MNs. SMN deficiency in postnatal MNs produces a depletion of functional CBs and relocalization of coilin, which is a scaffold protein of CBs, in snRNP-free perinucleolar caps or within the nucleolus. Disruption of CBs is the earliest nuclear sign of MN degeneration. We demonstrate that depletion of CBs, with loss of CB-nucleolus interactions, induces a progressive nucleolar dysfunction in ribosome biogenesis. It includes reorganization and loss of nucleolar transcription units, segregation of dense fibrillar and granular components, retention of SUMO-conjugated proteins in intranucleolar bodies and a reactive, compensatory, up-regulation of mature 18S rRNA and genes encoding key nucleolar proteins, such as upstream binding factor, fibrillarin, nucleolin and nucleophosmin. We propose that CB depletion and nucleolar alterations are essential components of the dysfunction of RNA metabolism in SMA.This work was supported by the following grants: “Dirección General de Investigación” of Spain (BFU2014-54754-P), “Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED; CB06/05/0037)” from Spain, and “Ministerio de Economía y Competitividad” of Spain cofinanced by FEDER (SAF2015-70801-R). Dr. Tapia is a recipient of a grant from SMA Europe cofinanced by FundAME (Spain) and "Instituto de Investigación Valdecilla" (IDIVAL; Next-Val) from Santander, Spain