Alternative polyadenylation (APA) of mRNAs has emerged as an important mechanism for post-transcriptional gene regulation in higher eukaryotes. Although microarrays have recently been used to characterize APA globally, they have a number of serious limitations that prevents comprehensive and highly quantitative analysis. To better characterize APA and its regulation, we have developed a deep sequencing-based method called Poly(A) Site Sequencing (PAS-Seq) for quantitatively profiling RNA polyadenylation at the transcriptome level. PAS-Seq not only accurately and comprehensively identifies poly(A) junctions in mRNAs and noncoding RNAs, but also provides quantitative information on the relative abundance of polyadenylated RNAs. PAS-Seq analyses of human and mouse transcriptomes showed that 40%–50% of all expressed genes produce alternatively polyadenylated mRNAs. Furthermore, our study detected evolutionarily conserved polyadenylation of histone mRNAs and revealed novel features of mitochondrial RNA polyadenylation. Finally, PAS-Seq analyses of mouse embryonic stem (ES) cells, neural stem/progenitor (NSP) cells, and neurons not only identified more poly(A) sites than what was found in the entire mouse EST database, but also detected significant changes in the global APA profile that lead to lengthening of 3′ untranslated regions (UTR) in many mRNAs during stem cell differentiation. Together, our PAS-Seq analyses revealed a complex landscape of RNA polyadenylation in mammalian cells and the dynamic regulation of APA during stem cell differentiation.

The splicing of pre‐mRNAs is an essential step of gene expression in eukaryotes. Introns are removed from split genes through the activities of the spliceosome, a large ribonuclear machine that is conserved throughout the eukaryotic lineage. While unicellular eukaryotes are characterized by less complex splicing, pre‐mRNA splicing of multicellular organisms is often associated with extensive alternative splicing that significantly enriches their proteome. The alternative selection of splice sites and exons permits multicellular organisms to modulate gene expression patterns in a cell type‐specific fashion, thus contributing to their functional diversification. Alternative splicing is a regulated process that is mainly influenced by the activities of splicing regulators, such as SR proteins or hnRNPs. These modular factors have evolved from a common ancestor through gene duplication events to a diverse group of splicing regulators that mediate exon recognition through their sequence‐specific binding to pre‐mRNAs. Given the strong correlations between intron expansion, the complexity of pre‐mRNA splicing, and the emergence of splicing regulators, it is argued that the increased presence of SR and hnRNP proteins promoted the evolution of alternative splicing through relaxation of the sequence requirements of splice junctions.WIREs RNA2012, 3:1–12. doi: 10.1002/wrna.100This article is categorized under: RNA Evolution and Genomics > RNA and Ribonucleoprotein Evolution RNA Processing > Splicing Regulation/Alternative Splicing

The processing of pre-mRNAs is a fundamental step required for the expression of most metazoan genes. Members of the family of serine/arginine (SR)-rich proteins are critical components of the machineries carrying out these essential processing events, highlighting their importance in maintaining efficient gene expression. SR proteins are characterized by their ability to interact simultaneously with RNA and other protein components via an RNA recognition motif (RRM) and through a domain rich in arginine and serine residues, the RS domain. Their functional roles in gene expression are surprisingly diverse, ranging from their classical involvement in constitutive and alternative pre-mRNA splicing to various post-splicing activities, including mRNA nuclear export, nonsense-mediated decay, and mRNA translation. These activities point up the importance of SR proteins during the regulation of mRNA metabolism.

The exon/intron architecture of genes determines whether components of the spliceosome recognize splice sites across the intron or across the exon. Using in vitro splicing assays, we demonstrate that splice-site recognition across introns ceases when intron size is between 200 and 250 nucleotides. Beyond this threshold, splice sites are recognized across the exon. Splice-site recognition across the intron is significantly more efficient than splice-site recognition across the exon, resulting in enhanced inclusion of exons with weak splice sites. Thus, intron size can profoundly influence the likelihood that an exon is constitutively or alternatively spliced. An EST-based alternative-splicing database was used to determine whether the exon/intron architecture influences the probability of alternative splicing in the Drosophila and human genomes. Drosophila exons flanked by long introns display an up to 90-fold-higher probability of being alternatively spliced compared with exons flanked by two short introns, demonstrating that the exon/intron architecture in Drosophila is a major determinant in governing the frequency of alternative splicing. Exon skipping is also more likely to occur when exons are flanked by long introns in the human genome. Interestingly, experimental and computational analyses show that the length of the upstream intron is more influential in inducing alternative splicing than is the length of the downstream intron. We conclude that the size and location of the flanking introns control the mechanism of splice-site recognition and influence the frequency and the type of alternative splicing that a pre-mRNA transcript undergoes.