Combinatorial control of exon recognition

Pre-mRNA splicing is a fundamental process required for the expression of most metazoan genes. It is carried out by the spliceosome, which catalyzes the removal of noncoding intronic sequences to assemble exons into mature mRNAs prior to export and translation. Given the complexity of higher eukaryotic genes and the relatively low level of splice site conservation, the precision of the splicing machinery in recognizing and pairing splice sites is impressive. Introns ranging in size from <100 up to 100,000 bases are removed efficiently. At the same time, a large number of alternative splicing events are observed between different cell types, during development, or during other biological processes. This extensive alternative splicing implies a significant flexibility of the spliceosome to identify and process exons within a given pre-mRNA. To reach this flexibility, splice site selection in higher eukaryotes has evolved to depend on multiple parameters such as splice site strength, the presence or absence of splicing regulators, RNA secondary structures, the exon/intron architecture, and the process of pre-mRNA synthesis itself. The relative contributions of each of these parameters control how efficiently splice sites are recognized and flanking introns are removed.     

Subdivision of large introns in Drosophila by recursive splicing at nonexonic elements

Many genes with important roles in development and disease contain exceptionally long introns, but special mechanisms for their expression have not been investigated. We present bioinformatic, phylogenetic, and experimental evidence in Drosophila for a mechanism that subdivides many large introns by recursive splicing at nonexonic elements and alternative exons. Recursive splice sites predicted with highly stringent criteria are found at much higher frequency than expected in the sense strands of introns >20 kb, but they are found only at the expected frequency on the antisense strands, and they are underrepresented within introns <10 kb. The predicted sites in long introns are highly conserved between Drosophila melanogaster and Drosophila pseudoobscura, despite extensive divergence of other sequences within the same introns. These patterns of enrichment and conservation indicate that recursive splice sites are advantageous in the context of long introns. Experimental analyses of in vivo processing intermediates and lariat products from four large introns in the unrelated genes kuzbanian, outspread, and Ultrabithorax confirmed that these introns are removed by a series of recursive splicing steps using the predicted nonexonic sites. Mutation of nonexonic site RP3 within Ultrabithorax also confirmed that recursive splicing is the predominant processing pathway even with a shortened version of the intron. We discuss currently known and potential roles for recursive splicing.     

Finding alternative splicing patterns with strong support from expressed sequences on individual exons/introns

We consider the problem of predicting alternative splicing patterns from a set of expressed sequences (cDNAs and ESTs). Some of these expressed sequences may be errorous, thus forming incorrect exons/introns. These incorrect exons/introns may cause a lot of false positives. For example, we examined a popular alternative splicing database, ECgene, which predicts alternate splicing patterns from expressed sequences. The result shows that about 81.3%-81.6% (sensitivity) of known patterns are found, but the specificity can be as low as 5.9%. Based on the idea that errorous sequences are usually not consistent with other sequences, in this paper we provide an alternative approach for finding alternative splicing patterns which ensures that individual exons/introns of the reported patterns have enough support from the expressed sequences. On the same dataset, our approach can achieve a much higher specificity and a slight increase in sensitivity (38.9% and 84.9%, respectively). Our approach also gives better results compared with popular alternative splicing databases (ASD, ECgene, SpliceNest) and the software ClusterMerge.     

ATP-dependent interaction of yeast U5 snRNA loop 1 with the 5' splice site.

Pre-messenger RNA splicing is a two-step process by which introns are removed and exons joined together. In yeast, the U5 snRNA loop 1 interacts with the 5' exon before the first step of splicing and with the 5' and 3' exons before the second step. In vitro studies revealed that yeast U5 loop 1 is not required for the first step of splicing but is essential for holding the 5' and 3' exons for ligation during the second step. It is critical, therefore, that loop 1 contacts the 5' exon before the first step of splicing to hold this exon following cleavage from the pre-mRNA. At present it is not known how U5 loop 1 is positioned on the 5' exon prior to the first step of splicing. To address this question, we have used site-specific photoactivated crosslinking in yeast spliceosomes to investigate the interaction of U5 loop 1 with the pre-mRNA prior to the first step of splicing. We have found that the highly conserved uridines in loop 1 make ATP-dependent contacts with an approximately 8-nt region at the 5' splice site that includes the invariant GU. These interactions are dependent on functional U2 and U6 snRNAs. Our results support a model where U5 snRNA loop 1 interacts with the 5' exon in two steps during its targeting to the 5' splice site.     

The RNA-binding protein hnRNPLL induces a T cell alternative splicing program delineated by differential intron retention in polyadenylated RNA

Background

Retention of a subset of introns in spliced polyadenylated mRNA is emerging as a frequent, unexplained finding from RNA deep sequencing in mammalian cells.

Results

Here we analyze intron retention in T lymphocytes by deep sequencing polyadenylated RNA. We show a developmentally regulated RNA-binding protein, hnRNPLL, induces retention of specific introns by sequencing RNA from T cells with an inactivating Hnrpll mutation and from B lymphocytes that physiologically downregulate Hnrpll during their differentiation. In Ptprc mRNA encoding the tyrosine phosphatase CD45, hnRNPLL induces selective retention of introns flanking exons 4 to 6; these correspond to the cassette exons containing hnRNPLL binding sites that are skipped in cells with normal, but not mutant or low, hnRNPLL. We identify similar patterns of hnRNPLL-induced differential intron retention flanking alternative exons in 14 other genes, representing novel elements of the hnRNPLL-induced splicing program in T cells. Retroviral expression of a normally spliced cDNA for one of these targets, Senp2, partially corrects the survival defect of Hnrpll-mutant T cells. We find that integrating a number of computational methods to detect genes with differentially retained introns provides a strategy to enrich for alternatively spliced exons in mammalian RNA-seq data, when complemented by RNA-seq analysis of purified cells with experimentally perturbed RNA-binding proteins.

Conclusions

Our findings demonstrate that intron retention in mRNA is induced by specific RNA-binding proteins and suggest a biological significance for this process in marking exons that are poised for alternative splicing.     

Sam68 regulates a set of alternatively spliced exons during neurogenesis

Sam68 (Src-associated in mitosis, 68 kDa) is a KH domain RNA binding protein implicated in a variety of cellular processes, including alternative pre-mRNA splicing, but its functions are not well understood. Using RNA interference knockdown of Sam68 expression and splicing-sensitive microarrays, we identified a set of alternative exons whose splicing depends on Sam68. Detailed analysis of one newly identified target exon in epsilon sarcoglycan (Sgce) showed that both RNA elements distributed across the adjacent introns and the RNA binding activity of Sam68 are necessary to repress the Sgce exon. Sam68 protein is upregulated upon neuronal differentiation of P19 cells, and many Sam68 RNA targets change in expression and splicing during this process. When Sam68 is knocked down by short hairpin RNAs, many Sam68-dependent splicing changes do not occur and P19 cells fail to differentiate. We also found that the differentiation of primary neuronal progenitor cells from embryonic mouse neocortex is suppressed by Sam68 depletion and promoted by Sam68 overexpression. Thus, Sam68 controls neurogenesis through its effects on a specific set of RNA targets.