Claudin-3 is a novel intestinal integrity marker in patients with alopecia areata: Correlation with the disease severity.

BACKGROUND: The development of alopecia areata is suggested to be influenced by intestinal permeability and gut dysbiosis. Claudin-3, an essential component of tight junctions which may act as an indicator of intestinal barrier integrity. AIMS: The study's objective was to evaluate the plasma concentration level of Claudin-3 in alopecia areata patients and its relationship to the severity of the condition. PATIENTS AND METHODS: In this case-control study, 50 alopecia areata patients and 30 healthy age and sex controls were involved. An enzyme-linked immunosorbent assay was used to determine the concentration of claudin-3 in the blood. RESULTS: Patients with alopecia areata had significantly higher plasma claudin-3 concentrations than healthy controls [median (interquartile range), 7.73 ng/ml (4.49-33.7) vs. 6.14 ng/ml (4.45-15.6), p < 0.005]. Positive relations were found between claudin-3 and SALT score (r = 0.675 & p-value < 0.001). CONCLUSIONS: Claudin-3, a gut permeability biomarker, is elevated in alopecia areata and correlates with disease severity.

iPSCs derived from insulin resistant offspring of type 2 diabetic patients show increased oxidative stress and lactate secretion.

BACKGROUND: The genetic factors associated with insulin resistance (IR) are not well understood. Clinical studies on first-degree relatives of type 2 diabetic (T2D) patients, which have the highest genetic predisposition to T2D, have given insights into the role of IR in T2D pathogenesis. Induced pluripotent stem cells (iPSCs) are excellent tools for disease modeling as they can retain the genetic imprint of the disease. Therefore, in this study, we aimed to investigate the genetic perturbations associated with insulin resistance (IR) in the offspring of T2D parents using patient-specific iPSCs. METHODS: We generated iPSCs from IR individuals (IR-iPSCs) that were offspring of T2D parents as well as from insulin-sensitive (IS-iPSCs) individuals. We then performed transcriptomics to identify key dysregulated gene networks in the IR-iPSCs in comparison to IS-iPSCs and functionally validated them. RESULTS: Transcriptomics on IR-iPSCs revealed dysregulated gene networks and biological processes indicating that they carry the genetic defects associated with IR that may lead to T2D. The IR-iPSCs had increased lactate secretion and a higher phosphorylation of AKT upon stimulation with insulin. IR-iPSCs have increased cellular oxidative stress indicated by a high production of reactive oxygen species and higher susceptibility to H2O2 -induced apoptosis. CONCLUSIONS: IR-iPSCs generated from offspring of diabetic patients confirm that oxidative stress and increased lactate secretion, associated with IR, are inherited in this population, and may place them at a high risk of T2D. Overall, our IR-iPSC model can be employed for T2D modeling and drug screening studies that target genetic perturbations associated with IR in individuals with a high risk for T2D.

Minor Intron Splicing from Basic Science to Disease.

Pre-mRNA splicing is an essential step in gene expression and is catalyzed by two machineries in eukaryotes: the major (U2 type) and minor (U12 type) spliceosomes. While the majority of introns in humans are U2 type, less than 0.4% are U12 type, also known as minor introns (mi-INTs), and require a specialized spliceosome composed of U11, U12, U4atac, U5, and U6atac snRNPs. The high evolutionary conservation and apparent splicing inefficiency of U12 introns have set them apart from their major counterparts and led to speculations on the purpose for their existence. However, recent studies challenged the simple concept of mi-INTs splicing inefficiency due to low abundance of their spliceosome and confirmed their regulatory role in alternative splicing, significantly impacting the expression of their host genes. Additionally, a growing list of minor spliceosome-associated diseases with tissue-specific pathologies affirmed the importance of minor splicing as a key regulatory pathway, which when deregulated could lead to tissue-specific pathologies due to specific alterations in the expression of some minor-intron-containing genes. Consequently, uncovering how mi-INTs splicing is regulated in a tissue-specific manner would allow for better understanding of disease pathogenesis and pave the way for novel therapies, which we highlight in this review.

Aberrant development of pancreatic beta cells derived from human iPSCs with FOXA2 deficiency.

FOXA2 has been identified as an essential factor for pancreas development and emerging evidence supports an association between FOXA2 and diabetes. Although the role of FOXA2 during pancreatic development is well-studied in animal models, its role during human islet cell development remains unclear. Here, we generated induced pluripotent stem cells (iPSCs) from a patient with FOXA2 haploinsufficiency (FOXA2+/- iPSCs) followed by beta-cell differentiation to understand the role of FOXA2 during pancreatic beta-cell development. Our results showed that FOXA2 haploinsufficiency resulted in aberrant expression of genes essential for the differentiation and proper functioning of beta cells. At pancreatic progenitor (PP2) and endocrine progenitor (EPs) stages, transcriptome analysis showed downregulation in genes associated with pancreatic development and diabetes and upregulation in genes associated with nervous system development and WNT signaling pathway. Knockout of FOXA2 in control iPSCs (FOXA2-/- iPSCs) led to severe phenotypes in EPs and beta-cell stages. The expression of NGN3 and its downstream targets at EPs as well as INSUILIN and GLUCAGON at the beta-cell stage, were almost absent in the cells derived from FOXA2-/- iPSCs. These findings indicate that FOXA2 is crucial for human pancreatic endocrine development and its defect may lead to diabetes based on FOXA2 dosage.

PDX1- /NKX6.1+ progenitors derived from human pluripotent stem cells as a novel source of insulin-secreting cells.

AIM: Beta cell replacement strategies are a promising alternative for diabetes treatment. Human pluripotent stem cells (hPSCs) serve as a scalable source for producing insulin-secreting cells for transplantation therapy. We recently generated novel hPSC-derived pancreatic progenitors, expressing high levels of the transcription factor NKX6.1, in the absence of PDX1 (PDX1- /NKX6.1+ ). Herein, our aim was to characterize this novel population and assess its ability to differentiate into insulin-secreting beta cells in vitro. MATERIALS AND METHODS: Three different hPSC lines were differentiated into PDX1- /NKX6.1+ progenitors, which were further differentiated into insulin-secreting cells using two different protocols. The progenitors and beta cells were extensively characterized. Transcriptome analysis was performed at different stages and compared with the profiles of various pancreatic counterparts. RESULTS: PDX1- /NKX6.1+ progenitors expressed high levels of nestin, a key marker of pancreatic islet-derived progenitors, in the absence of E-cadherin, similar to pancreatic mesenchymal stem cells. At progenitor stage, comparison of the two populations showed downregulation of pancreatic epithelial genes and upregulation of neuronal development genes in PDX1- /NKX6.1+ cells in comparison to the PDX1+ /NKX6.1+ cells. Interestingly, on further differentiation, PDX1- /NKX6.1+ cells generated mono-hormonal insulin+ cells and activated pancreatic key genes, such as PDX1. The transcriptome profile of PDX1- /NKX6.1+ -derived beta (3D-beta) was closely similar to those of human pancreatic islets and purified hPSC-derived beta cells. Also, the 3D-beta cells secreted C-peptide in response to increased glucose concentrations indicating their functionality. CONCLUSION: These findings provide a novel source of insulin-secreting cells that can be used for beta cell therapy for diabetes.

Scalable Generation of Mesenchymal Stem Cells and Adipocytes from Human Pluripotent Stem Cells.

Human pluripotent stem cells (hPSCs) can provide unlimited supply for mesenchymal stem cells (MSCs) and adipocytes that can be used for therapeutic applications. Here we developed a simple and highly efficient all-trans-retinoic acid (RA)-based method for generating an off-the-shelf and scalable number of human pluripotent stem cell (hPSC)-derived MSCs with enhanced adipogenic potential. We showed that short exposure of multiple hPSC lines (hESCs/hiPSCs) to 10 µM RA dramatically enhances embryoid body (EB) formation through regulation of genes activating signaling pathways associated with cell proliferation, survival and adhesion, among others. Disruption of cell adhesion induced the subsequent differentiation of the highly expanded RA-derived EB-forming cells into a pure population of multipotent MSCs (up to 1542-fold increase in comparison to RA-untreated counterparts). Interestingly, the RA-derived MSCs displayed enhanced differentiation potential into adipocytes. Thus, these findings present a novel RA-based approach for providing an unlimited source of MSCs and adipocytes that can be used for regenerative medicine, drug screening and disease modeling applications.

Keratinocytes Derived from Patient-Specific Induced Pluripotent Stem Cells Recapitulate the Genetic Signature of Psoriasis Disease.

Psoriasis is characterized by hyperproliferation and defective differentiation of keratinocytes (KCs). Patients with psoriasis are at a high risk of developing diabetes and cardiovascular diseases. The debate on the genetic origin of psoriasis pathogenesis remains unresolved due to lack of suitable in vitro human models mimicking the disease phenotypes. In this study, we provide the first human induced pluripotent stem cell (iPSC) model for psoriasis carrying the genetic signature of the patients. iPSCs were generated from patients with psoriasis (PsO-iPSCs) and healthy donors (Ctr-iPSCs) and were efficiently differentiated into mature KCs. RNA sequencing of KCs derived from Ctr-iPSCs and PsO-iPSCs identified 361 commonly upregulated and 412 commonly downregulated genes. KCs derived from PsO-iPSCs showed dysregulated transcripts associated with psoriasis and KC differentiation, such as HLA-C, KLF4, chemokines, type I interferon-inducible genes, solute carrier family, IVL, DSG1, and HLA-DQA1, as well as transcripts associated with insulin resistance, such as IRS2, GDF15, GLUT10, and GLUT14. Our data suggest that the KC abnormalities are the main driver triggering psoriasis pathology and highlights the substantial contribution of genetic predisposition in the development of psoriasis and insulin resistance.

U1 snRNP regulates cancer cell migration and invasion in vitro.

Stimulated cells and cancer cells have widespread shortening of mRNA 3'-untranslated regions (3'UTRs) and switches to shorter mRNA isoforms due to usage of more proximal polyadenylation signals (PASs) in introns and last exons. U1 snRNP (U1), vertebrates' most abundant non-coding (spliceosomal) small nuclear RNA, silences proximal PASs and its inhibition with antisense morpholino oligonucleotides (U1 AMO) triggers widespread premature transcription termination and mRNA shortening. Here we show that low U1 AMO doses increase cancer cells' migration and invasion in vitro by up to 500%, whereas U1 over-expression has the opposite effect. In addition to 3'UTR length, numerous transcriptome changes that could contribute to this phenotype are observed, including alternative splicing, and mRNA expression levels of proto-oncogenes and tumor suppressors. These findings reveal an unexpected role for U1 homeostasis (available U1 relative to transcription) in oncogenic and activated cell states, and suggest U1 as a potential target for their modulation.

The Cancer Spliceome: Reprograming of Alternative Splicing in Cancer.

Alternative splicing allows for the expression of multiple RNA and protein isoforms from one gene, making it a major contributor to transcriptome and proteome diversification in eukaryotes. Advances in next generation sequencing technologies and genome-wide analyses have recently underscored the fact that the vast majority of multi-exon genes under normal physiology engage in alternative splicing in tissue-specific and developmental-specific manner. On the other hand, cancer cells exhibit remarkable transcriptome alterations partly by adopting cancer-specific splicing isoforms. These isoforms and their encoded proteins are not insignificant byproducts of the abnormal physiology of cancer cells, but either drivers of cancer progression or small but significant contributors to specific cancer hallmarks. Thus, it is paramount that the pathways that regulate alternative splicing in cancer, including the splicing factors that bind to pre-mRNAs and modulate spliceosome recruitment. In this review, we present a few distinct cases of alternative splicing in cancer, with an emphasis on their regulation as well as their contribution to cancer cell phenotype. Several categories of splicing aberrations are highlighted, including alterations in cancer-related genes that directly affect their pre-mRNA splicing, mutations in genes encoding splicing factors or core spliceosomal subunits, and the seemingly mutation-free disruptions in the balance of the expression of RNA-binding proteins, including components of both the major (U2-dependent) and minor (U12-dependent) spliceosomes. Given that the latter two classes cause global alterations in splicing that affect a wide range of genes, it remains a challenge to identify the ones that contribute to cancer progression. These challenges necessitate a systematic approach to decipher these aberrations and their impact on cancer. Ultimately, a sufficient understanding of splicing deregulation in cancer is predicted to pave the way for novel and innovative RNA-based therapies.

U1 snRNP telescripting regulates a size-function-stratified human genome.

U1 snRNP (U1) functions in splicing introns and telescripting, which suppresses premature cleavage and polyadenylation (PCPA). Using U1 inhibition in human cells, we show that U1 telescripting is selectively required for sustaining long-distance transcription elongation in introns of large genes (median 39 kb). Evidence of widespread PCPA in the same locations in normal tissues reveals that large genes incur natural transcription attrition. Underscoring the importance of U1 telescripting as a gene-size-based mRNA-regulation mechanism, small genes were not sensitive to PCPA, and the spliced-mRNA productivity of ∼1,000 small genes (median 6.8 kb) increased upon U1 inhibition. Notably, these small, upregulated genes were enriched in functions related to acute stimuli and cell-survival response, whereas genes subject to PCPA were enriched in cell-cycle progression and developmental functions. This gene size-function polarization increased in metazoan evolution by enormous intron expansion. We propose that telescripting adds an overarching layer of regulation to size-function-stratified genomes, leveraged by selective intron expansion to rapidly shift gene expression priorities.

A U1 snRNP-specific assembly pathway reveals the SMN complex as a versatile hub for RNP exchange.

Despite equal snRNP stoichiometry in spliceosomes, U1 snRNP (U1) is typically the most abundant vertebrate snRNP. Mechanisms regulating U1 overabundance and snRNP repertoire are unknown. In Sm-core assembly, a key snRNP-biogenesis step mediated by the SMN complex, the snRNA-specific RNA-binding protein (RBP) Gemin5 delivers pre-snRNAs, which join SMN-Gemin2-recruited Sm proteins. We show that the human U1-specific RBP U1-70K can bridge pre-U1 to SMN-Gemin2-Sm, in a Gemin5-independent manner, thus establishing an additional and U1-exclusive Sm core-assembly pathway. U1-70K hijacks SMN-Gemin2-Sm, enhancing Sm-core assembly on U1s and inhibiting that on other snRNAs, thereby promoting U1 overabundance and regulating snRNP repertoire. SMN-Gemin2's ability to facilitate transactions between different RBPs and RNAs explains its multi-RBP valency and the myriad transcriptome perturbations associated with SMN deficiency in neurodegenerative spinal muscular atrophy. We propose that SMN-Gemin2 is a versatile hub for RNP exchange that functions broadly in RNA metabolism.

Minor introns are embedded molecular switches regulated by highly unstable U6atac snRNA.

Eukaryotes have two types of spliceosomes, comprised of either major (U1, U2, U4, U5, U6) or minor (U11, U12, U4atac, U6atac; <1%) snRNPs. The high conservation of minor introns, typically one amidst many major introns in several hundred genes, despite their poor splicing, has been a long-standing enigma. Here, we discovered that the low abundance minor spliceosome's catalytic snRNP, U6atac, is strikingly unstable (t½<2 hr). We show that U6atac level depends on both RNA polymerases II and III and can be rapidly increased by cell stress-activated kinase p38MAPK, which stabilizes it, enhancing mRNA expression of hundreds of minor intron-containing genes that are otherwise suppressed by limiting U6atac. Furthermore, p38MAPK-dependent U6atac modulation can control minor intron-containing tumor suppressor PTEN expression and cytokine production. We propose that minor introns are embedded molecular switches regulated by U6atac abundance, providing a novel post-transcriptional gene expression mechanism and a rationale for the minor spliceosome's evolutionary conservation. DOI:http://dx.doi.org/10.7554/eLife.00780.001.

U1 snRNP determines mRNA length and regulates isoform expression.

U1 snRNP (U1), in addition to its splicing role, protects pre-mRNAs from drastic premature termination by cleavage and polyadenylation (PCPA) at cryptic polyadenylation signals (PASs) in introns. Here, a high-throughput sequencing strategy of differentially expressed transcripts (HIDE-seq) mapped PCPA sites genome wide in divergent organisms. Surprisingly, whereas U1 depletion terminated most nascent gene transcripts within ~1 kb, moderate functional U1 level decreases, insufficient to inhibit splicing, dose-dependently shifted PCPA downstream and elicited mRNA 3' UTR shortening and proximal 3' exon switching characteristic of activated immune and neuronal cells, stem cells, and cancer. Activated neurons' signature mRNA shortening could be recapitulated by U1 decrease and antagonized by U1 overexpression. Importantly, we show that rapid and transient transcriptional upregulation inherent to neuronal activation physiology creates U1 shortage relative to pre-mRNAs. Additional experiments suggest cotranscriptional PCPA counteracted by U1 association with nascent transcripts, a process we term telescripting, ensuring transcriptome integrity and regulating mRNA length.

A quantitative high-throughput in vitro splicing assay identifies inhibitors of spliceosome catalysis.

Despite intensive research, there are very few reagents with which to modulate and dissect the mRNA splicing pathway. Here, we describe a novel approach to identify such tools, based on detection of the exon junction complex (EJC), a unique molecular signature that splicing leaves on mRNAs. We developed a high-throughput, splicing-dependent EJC immunoprecipitation (EJIPT) assay to quantitate mRNAs spliced from biotin-tagged pre-mRNAs in cell extracts, using antibodies to EJC components Y14 and eukaryotic translation initiation factor 4aIII (eIF4AIII). Deploying EJIPT we performed high-throughput screening (HTS) in conjunction with secondary assays to identify splicing inhibitors. We describe the identification of 1,4-naphthoquinones and 1,4-heterocyclic quinones with known anticancer activity as potent and selective splicing inhibitors. Interestingly, and unlike previously described small molecules, most of which target early steps, our inhibitors represented by the benzothiazole-4,7-dione, BN82685, block the second of two trans-esterification reactions in splicing, preventing the release of intron lariat and ligation of exons. We show that BN82685 inhibits activated spliceosomes' elaborate structural rearrangements that are required for second-step catalysis, allowing definition of spliceosomes stalled in midcatalysis. EJIPT provides a platform for characterization and discovery of splicing and EJC modulators.

U1 snRNP protects pre-mRNAs from premature cleavage and polyadenylation.

In eukaryotes, U1 small nuclear ribonucleoprotein (snRNP) forms spliceosomes in equal stoichiometry with U2, U4, U5 and U6 snRNPs; however, its abundance in human far exceeds that of the other snRNPs. Here we used antisense morpholino oligonucleotide to U1 snRNA to achieve functional U1 snRNP knockdown in HeLa cells, and identified accumulated unspliced pre-mRNAs by genomic tiling microarrays. In addition to inhibiting splicing, U1 snRNP knockdown caused premature cleavage and polyadenylation in numerous pre-mRNAs at cryptic polyadenylation signals, frequently in introns near (<5 kilobases) the start of the transcript. This did not occur when splicing was inhibited with U2 snRNA antisense morpholino oligonucleotide or the U2-snRNP-inactivating drug spliceostatin A unless U1 antisense morpholino oligonucleotide was also included. We further show that U1 snRNA-pre-mRNA base pairing was required to suppress premature cleavage and polyadenylation from nearby cryptic polyadenylation signals located in introns. These findings reveal a critical splicing-independent function for U1 snRNP in protecting the transcriptome, which we propose explains its overabundance.

Rapid-response splicing reporter screens identify differential regulators of constitutive and alternative splicing.

Bioactive compounds have been invaluable for dissecting the mechanisms, regulation, and functions of cellular processes. However, very few such reagents have been described for pre-mRNA splicing. To facilitate their systematic discovery, we developed a high-throughput cell-based assay that measures pre-mRNA splicing by utilizing a quantitative reporter system with advantageous features. The reporter, consisting of a destabilized, intron-containing luciferase expressed from a short-lived mRNA, allows rapid screens (<4 h), thereby obviating the potential toxicity of splicing inhibitors. We describe three inhibitors (out of >23,000 screened), all pharmacologically active: clotrimazole, flunarizine, and chlorhexidine. Interestingly, none was a general splicing inhibitor. Rather, each caused distinct splicing changes of numerous genes. We further discovered the target of action of chlorhexidine and show that it is a selective inhibitor of specific Cdc2-like kinases (Clks) that phosphorylate serine-arginine-rich (SR) protein splicing factors. Our findings reveal unexpected activities of clinically used drugs in splicing and uncover differential regulation of constitutively spliced introns.

Human T-cell leukemia virus type 2 Rex carboxy terminus is an inhibitory/stability domain that regulates Rex functional activity and viral replication.

Human T-cell leukemia virus (HTLV) regulatory protein, Rex, functions to increase the expression of the viral structural and enzymatic gene products. The phosphorylation of two serine residues (S151 and S153) at the C terminus is important for the function of HTLV-2 Rex (Rex-2). The Rex-2 phosphomimetic double mutant (S151D, S153D) is locked in a functionally active conformation. Since rex and tax genes overlap, Rex S151D and S153D mutants were found to alter the Tax oncoprotein coding sequence and transactivation activities. Therefore, additional Rex-2 mutants including P152D, A157D, S151Term, and S158Term were generated and characterized ("Term" indicates termination codon). All Rex-2 mutants and wild-type (wt) Rex-2 localized predominantly to the nucleus/nucleolus, but in contrast to the detection of phosphorylated and unphosphorylated forms of wt Rex-2 (p26 and p24), mutant proteins were detected as a single phosphoprotein species. We found that Rex P152D, A157D, and S158Term mutants are more functionally active than wt Rex-2 and that the Rex-2 C terminus and its specific phosphorylation state are required for stability and optimal expression. In the context of the provirus, the more active Rex mutants (A157D or S158Term) promoted increased viral protein production, increased viral infectious spread, and enhanced HTLV-2-mediated cellular proliferation. Moreover, these Rex mutant viruses replicated and persisted in inoculated rabbits despite higher antiviral antibody responses. Thus, we identified in Rex-2 a novel C-terminal inhibitory domain that regulates functional activity and is positively regulated through phosphorylation. The ability of this domain to modulate viral replication likely plays a key role in the infectious spread of the virus and in virus-induced cellular proliferation.

Human T-cell leukemia virus type 2 post-transcriptional control protein p28 is required for viral infectivity and persistence in vivo.

BACKGROUND: Human T-cell leukemia virus (HTLV) type 1 and type 2 are related but distinct pathogenic complex retroviruses. HTLV-1 is associated with adult T-cell leukemia and a variety of immune-mediated disorders including the chronic neurological disease termed HTLV-1-associated myelopathy/tropical spastic paraparesis. In contrast, HTLV-2 displays distinct biological differences and is much less pathogenic, with only a few reported cases of leukemia and neurological disease associated with infection. In addition to the structural and enzymatic proteins, HTLV encodes regulatory (Tax and Rex) and accessory proteins. Tax and Rex positively regulate virus production and are critical for efficient viral replication and pathogenesis. Using an over-expression system approach, we recently reported that the accessory gene product of the HTLV-1 and HTLV-2 open reading frame (ORF) II (p30 and p28, respectively) acts as a negative regulator of both Tax and Rex by binding to and retaining their mRNA in the nucleus, leading to reduced protein expression and virion production. Further characterization revealed that p28 was distinct from p30 in that it was devoid of major transcriptional modulating activity, suggesting potentially divergent functions that may be responsible for the distinct pathobiologies of HTLV-1 and HTLV-2. RESULTS: In this study, we investigated the functional significance of p28 in HTLV-2 infection, proliferation, and immortaliztion of primary T-cells in culture, and viral survival in an infectious rabbit animal model. An HTLV-2 p28 knockout virus (HTLV-2Deltap28) was generated and evaluated. Infectivity and immortalization capacity of HTLV-2Deltap28 in vitro was indistinguishable from wild type HTLV-2. In contrast, we showed that viral replication was severely attenuated in rabbits inoculated with HTLV-2Deltap28 and the mutant virus failed to establish persistent infection. CONCLUSION: We provide direct evidence that p28 is dispensable for viral replication and cellular immortalization of primary T-lymphocytes in cell culture. However, our data indicate that p28 function is critical for viral survival in vivo. Our results are consistent with the hypothesis that p28 repression of Tax and Rex-mediated viral gene expression may facilitate survival of these cells by down-modulating overall viral gene expression.

SMN deficiency causes tissue-specific perturbations in the repertoire of snRNAs and widespread defects in splicing.

The survival of motor neurons (SMN) protein is essential for the biogenesis of small nuclear RNA (snRNA)-ribonucleoproteins (snRNPs), the major components of the pre-mRNA splicing machinery. Though it is ubiquitously expressed, SMN deficiency causes the motor neuron degenerative disease spinal muscular atrophy (SMA). We show here that SMN deficiency, similar to that which occurs in severe SMA, has unexpected cell type-specific effects on the repertoire of snRNAs and mRNAs. It alters the stoichiometry of snRNAs and causes widespread pre-mRNA splicing defects in numerous transcripts of diverse genes, preferentially those containing a large number of introns, in SMN-deficient mouse tissues. These findings reveal a key role for the SMN complex in RNA metabolism and in splicing regulation and indicate that SMA is a general splicing disease that is not restricted to motor neurons.

PYM binds the cytoplasmic exon-junction complex and ribosomes to enhance translation of spliced mRNAs.

Messenger RNAs produced by splicing are translated more efficiently than those produced from similar intronless precursor mRNAs (pre-mRNAs). The exon-junction complex (EJC) probably mediates this enhancement; however, the specific link between the EJC and the translation machinery has not been identified. The EJC proteins Y14 and magoh remain bound to spliced mRNAs after their export from the nucleus to the cytoplasm and are removed only when these mRNAs are translated. Here we show that PYM, a 29-kDa protein that binds the Y14-magoh complex in the cytoplasm, also binds, via a separate domain, to the small (40S) ribosomal subunit and the 48S preinitiation complex. Furthermore, PYM knockdown reduces the translation efficiency of a reporter protein produced from intron-containing, but not intronless, pre-mRNA. We suggest that PYM functions as a bridge between EJC-bearing spliced mRNAs and the translation machinery to enhance translation of the mRNAs.