Alternate mRNA splicing is required for synthesis of adeno-associated virus VP1 capsid protein.

Fine-structure mapping of the capsid-specific mRNAs from adeno-associated virus (AAV) revealed an alternate splicing pattern in these RNAs. S1 nuclease and primer extension analyses showed that splicing of these mRNAs occurs at acceptor sites at nucleotide 2228 (major splice) or 2201 (minor splice). Both splice acceptors were ligated to the same 55-nucleotide leader in mature mRNAs. Both species were present in equal amounts in mRNA derived from AAV plasmid-transfected cells. However, when adenovirus infection accompanied the DNA transfection, the major splice predominated over the minor splice. Using cDNA clones of both the major and minor spliced mRNAs, we demonstrated that the largest AAV capsid protein, VP1, was derived from the minor spliced mRNA. The other capsid proteins, VP2 and VP3, came predominantly from the major spliced mRNA. These results, which describe the previously undetected minor splice, provide a mechanism for the production of all three AAV virion proteins.     

Glucose dissociates DDX21 dimers to regulate mRNA splicing and tissue differentiation

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MicroRNAs act complementarily to regulate disease-related mRNA modules in human diseases

MicroRNAs (miRNAs) play a key role in regulating mRNA expression, and individual miRNAs have been proposed as diagnostic and therapeutic candidates. The identification of such candidates is complicated by the involvement of multiple miRNAs and mRNAs as well as unknown disease topology of the miRNAs. Here, we investigated if disease-associated miRNAs regulate modules of disease-associated mRNAs, if those miRNAs act complementarily or synergistically, and if single or combinations of miRNAs can be targeted to alter module functions. We first analyzed publicly available miRNA and mRNA expression data for five different diseases. Integrated target prediction and network-based analysis showed that the miRNAs regulated modules of disease-relevant genes. Most of the miRNAs acted complementarily to regulate multiple mRNAs. To functionally test these findings, we repeated the analysis using our own miRNA and mRNA expression data from CD4+ T cells from patients with seasonal allergic rhinitis. This is a good model of complex diseases because of its well-defined phenotype and pathogenesis. Combined computational and functional studies confirmed that miRNAs mainly acted complementarily and that a combination of two complementary miRNAs, miR-223 and miR-139-3p, could be targeted to alter disease-relevant module functions, namely, the release of type 2 helper T-cell (Th2) cytokines. Taken together, our findings indicate that miRNAs act complementarily to regulate modules of disease-related mRNAs and can be targeted to alter disease-relevant functions.     

Noisy splicing drives mRNA isoform diversity in human cells

While the majority of multiexonic human genes show some evidence of alternative splicing, it is unclear what fraction of observed splice forms is functionally relevant. In this study, we examine the extent of alternative splicing in human cells using deep RNA sequencing and de novo identification of splice junctions. We demonstrate the existence of a large class of low abundance isoforms, encompassing approximately 150,000 previously unannotated splice junctions in our data. Newly-identified splice sites show little evidence of evolutionary conservation, suggesting that the majority are due to erroneous splice site choice. We show that sequence motifs involved in the recognition of exons are enriched in the vicinity of unconserved splice sites. We estimate that the average intron has a splicing error rate of approximately 0.7% and show that introns in highly expressed genes are spliced more accurately, likely due to their shorter length. These results implicate noisy splicing as an important property of genome evolution.     

Immunologic and physicochemical evidence for conformational changes occurring on conversion of human mast cell tryptase from active tetramer to inactive monomer. Production of monoclonal antibodies recognizing active tryptase

The catalytic activity of human tryptase, a mast cell neutral endoprotease, is expressed when the enzyme is in its tetrameric form, but is lost under physiologic conditions concomitant with a quaternary structural alteration involving conversion to a monomeric form. The associated changes in the CD spectra noted in the current study indicate accompanying alterations in the secondary structure of the protein. In particular, the progressive disappearance of the negative minimum centered at 228 nm suggests an effect on beta-sheet structure, which may be important for monomer-monomer interaction and/or stabilization of catalytic activity. Dextran sulfate, like heparin, stabilizes the catalytic activity and quaternary structure of tryptase and also maintains the native secondary structure of the enzyme at and beyond a temperature of 40 degrees C. Dextran sulfate-stabilized tryptase therefore was used as an immunogen to which were produced three murine mAb (B2, C11, and G4) recognizing the catalytically active form of the enzyme. Inactive tryptase bound to plastic microtiter wells was not recognized by any of the newly made antibodies, whereas inactive tryptase in solution was recognized by G4, which when biotinylated, could be used as a detector antibody in a sandwich ELISA for tryptase. Each of the newly made mAb recognized the catalytically active form of tryptase. Thus, alterations in epitopes, perhaps reflecting tertiary structural alterations as well as changes in secondary and quaternary conformations, occur with tryptase inactivation. A pragmatic result of these newly generated antibodies is the affinity purification to homogeneity of active tryptase by sequential chromatography with B2 coupled to CH-Sepharose and heparin-agarose. Tryptase purified by this technique had a specific activity with p-tosyl-L-arginine methyl ester of 117 +/- 9 U/mg and had 3.9 +/- 0.3 active sites per molecule of active enzyme (134,000 m.w.) as titrated with p-nitrophenyl-p'-guanidinobenzoate. The spectral and immunologic data in the current study are consistent with concerted conformational alterations in the secondary and tertiary as well as quaternary structures of tryptase associated with loss of catalytic activity. Failure to reverse any of these alterations with dextran sulfate suggests that the pathway of tetramer assembly in vivo is more complicated than simple subunit association.     

Sororin pre‐mRNA splicing is required for proper sister chromatid cohesion in human cells

Sister chromatid cohesion, which depends on cohesin, is essential for the faithful segregation of replicated chromosomes. Here, we report that splicing complex Prp19 is essential for cohesion in both G2 and mitosis, and consequently for the proper progression of the cell through mitosis. Inactivation of splicing factors SF3a120 and U2AF65 induces similar cohesion defects to Prp19 complex inactivation. Our data indicate that these splicing factors are all required for the accumulation of cohesion factor Sororin, by facilitating the proper splicing of its pre‐mRNA. Finally, we show that ectopic expression of Sororin corrects defective cohesion caused by Prp19 complex inactivation. We propose that the Prp19 complex and the splicing machinery contribute to the establishment of cohesion by promoting Sororin accumulation during S phase, and are, therefore, essential to the maintenance of genome stability.     

Distribution of exonic splicing enhancer elements in human genes

Ab initio prediction of functional exon splicing enhancer (ESE) elements based on RNA sequences present a challenge in the evaluation of the functional impacts of human genetic polymorphisms on splicing. To better understand the behavior of ESEs, we studied their distribution in human exons and introns for four known SR protein-binding motifs: SF2/SAF, SC35, SRp40, and SRp55. ESEs are enriched in regions in exons that are close to the splice sites, especially in the region 80 to 120 bases away from the ends of splice acceptor sites. Significant enrichment of ESEs is associated with weak splice acceptor sites but not weak donor sites. ESE density decreases at the 3 ends of long exons. ESEs are also enriched in introns with weak donor or acceptor sites. These characteristics of ESEs may help to predict functional ESE sites in RNA sequences.     

Rare variants regulate expression of nearby individual genes in multiple tissues

The rapid decrease in sequencing cost has enabled genetic studies to discover rare variants associated with complex diseases and traits. Once this association is identified, the next step is to understand the genetic mechanism of rare variants on how the variants influence diseases. Similar to the hypothesis of common variants, rare variants may affect diseases by regulating gene expression, and recently, several studies have identified the effects of rare variants on gene expression using heritability and expression outlier analyses. However, identifying individual genes whose expression is regulated by rare variants has been challenging due to the relatively small sample size of expression quantitative trait loci studies and statistical approaches not optimized to detect the effects of rare variants. In this study, we analyze whole-genome sequencing and RNA-seq data of 681 European individuals collected for the Genotype-Tissue Expression (GTEx) project (v8) to identify individual genes in 49 human tissues whose expression is regulated by rare variants. To improve statistical power, we develop an approach based on a likelihood ratio test that combines effects of multiple rare variants in a nonlinear manner and has higher power than previous approaches. Using GTEx data, we identify many genes regulated by rare variants, and some of them are only regulated by rare variants and not by common variants. We also find that genes regulated by rare variants are enriched for expression outliers and disease-causing genes. These results suggest the regulatory effects of rare variants, which would be important in interpreting associations of rare variants with complex traits.     

UBL5 is essential for pre‐mRNA splicing and sister chromatid cohesion in human cells

UBL5 is an atypical ubiquitin‐like protein, whose function in metazoans remains largely unexplored. We show that UBL5 is required for sister chromatid cohesion maintenance in human cells. UBL5 primarily associates with spliceosomal proteins, and UBL5 depletion decreases pre‐mRNA splicing efficiency, leading to globally enhanced intron retention. Defective sister chromatid cohesion is a general consequence of dysfunctional pre‐mRNA splicing, resulting from the selective downregulation of the cohesion protection factor Sororin. As the UBL5 yeast orthologue, Hub1, also promotes spliceosome functions, our results show that UBL5 plays an evolutionary conserved role in pre‐mRNA splicing, the integrity of which is essential for the fidelity of chromosome segregation.