The three major capsid proteins of adeno-

The three adeno-associated virus type 2 (AAV2) structural proteins (A, B, and C) are specified by transcripts generated from the most-rightward promoter (p40). Protein C (60 kilodaltons [kDa]), the most abundantly produced, is entirely contained within B (72 kDa) which, in turn, is contained within A (90 kDa). Although neither of the known structures of p40 transcripts, an unspliced 2.6-kilobase (kb) RNA and a spliced 2.3-kb RNA, possesses an AUG-initiated open reading frame that accounts for the synthesis of proteins A and B, recent evidence indicates that B is initiated by a unique eucaryotic initiation codon (ACG) (S. P. Becerra, J. A.

The first stable complex formed during the assembly of spliceosomes onto pre-mRNA substrates in mammals includes U1 snRNP, which recognizes the 5 ′ splice site, and the splicing factors SF1 and U2AF, which bind the branch point sequence, polypyrimidine tract, and 3 ′ splice site. The 5 ′ and 3 ′ splice site complexes are thought to be joined together by proteinprotein interactions mediated by factors that ensure the fidelity of the initial splice site recognition. In this study, we identified and characterized PRPF40B, a putative mammalian ortholog of the U1 snRNP-associated yeast splicing factor Prp40. PRPF40B is highly enriched in speckles with a behavior similar to splicing factors. We demonstrated that PRPF40B interacts directly with SF1 and associates with U2AF 65 . Accordingly, PRPF40B colocalizes with these splicing factors in the cell nucleus. Splicing assays with reporter minigenes revealed that PRPF40B modulates alternative splice site selection. In the case of Fas regulation of alternative splicing, weak 5 ′ and 3 ′ splice sites and exonic sequences are required for PRPF40B function. Placing our data in a functional context, we also show that PRPF40B depletion increased Fas/CD95 receptor number and cell apoptosis, which suggests the ability of PRPF40B to alter the alternative splicing of key apoptotic genes to regulate cell survival.

The tightly regulated process of precursor messenger RNA (pre-mRNA) alternative splicing is a key mechanism to increase the number and complexity of proteins encoded by the genome. Evidence gathered in recent years has established that transcription and splicing are physically and functionally coupled and that this coupling may be an essential aspect of the regulation of splicing and alternative splicing. Recent advances in our understanding of transcription and of splicing regulation have uncovered the multiple interactions between components from both types of machinery. These interactions help to explain the functional coupling of RNAPII transcription and pre-mRNA alternative splicing for efficient and regulated gene expression at the molecular level. Recent technological advances, in addition to novel cell and molecular biology approaches, have led to the development of new tools for addressing mechanistic questions to achieve an integrated and global understanding of the functional coupling of RNAPII transcription and pre-mRNA alternative splicing. Here, we review major milestones and insights into RNA polymerase II transcription and pre-mRNA alternative splicing as well as new concepts and challenges that have arisen from multiple genome-wide approaches and analyses at the single-cell resolution.

The alternative splicing (AS) of precursor messenger RNA (pre-mRNA) is a tightly regulated process through which introns are removed to leave the resulting exons in the mRNA appropriately aligned and ligated. The AS of pre-mRNA is a key mechanism for increasing the complexity of proteins encoded in the genome. In humans, more than 90% of genes undergo AS, underscoring the importance of this process in RNA biogenesis. As such, AS misregulation underlies multiple human diseases. The splicing reaction is catalyzed by the spliceosome, a highly dynamic complex that assembles at or near the intron/exon boundaries and undergoes sequential conformational and compositional changes during splicing. The initial recognition of splice sites defines the exons that are going to be removed, which is a critical step in the highly regulated splicing process. Although the available lines of evidence are increasing, the molecular mechanisms governing AS, including the initial interactions occurring at intron/exon boundaries, and the factors that modulate these critical connections by functioning as a scaffold for active-site RNAs or proteins, remain poorly understood. In this review, we summarize the major hallmarks of the initial steps in the splicing process and the role of auxiliary factors that contribute to the assembly of the spliceosomal complex. We also discuss the role of the essential yeast Prp40 protein and its mammalian homologs in the specificity of this pre-mRNA processing event. In addition, we provide the first exhaustive phylogenetic analysis of the molecular evolution of Prp40 family members. WIREs RNA 2016, 7:17-32. doi: 10.1002/wrna.1312 For further resources related to this article, please visit the WIREs website.