Summary SR proteins have been studied extensively as a family of RNA binding proteins that participate in both constitutive and regulated pre-mRNA splicing in mammalian cells. However, SR proteins were first discovered as factors that interact with transcriptionally active chromatin. Recent studies have now uncovered properties that connect these once apparently disparate functions, showing that a subset of SR proteins seem to bind directly to the histone 3 tail, play an active role in transcriptional elongation, and co-localize with genes that are engaged in specific intra- and inter-chromosome interactions for coordinated regulation of gene expression in the nucleus. These transcription-related activities are also coupled with a further expansion of putative functions of specific SR protein family members in RNA metabolism downstream of mRNA splicing, from RNA export to stability control to translation. These findings therefore highlight the broader roles of SR proteins in vertical integration of gene expression and provide mechanistic insights into their contributions to genome stability and proper cell cycle progression in higher eukaryotic cells.

We have used nucleobase substitution and kinetic analysis to test the hypothesis that hammerhead catalysis occurs by a general acid-base mechanism, in which nucleobases are directly involved in deprotonation of the attacking 2'-hydroxyl group and protonation of the 5'-oxygen that serves as the leaving group in the cleavage reaction. We demonstrate that simultaneous substitution of two important nucleobases, G8 and G12, with 2,6-diaminopurine shifts the pH optimum of the cleavage reaction from greater than 9.5 to approximately 6.8 in two different hammerhead constructs. Controls involving substitution with other nucleobases and combinations of nucleobases at G5, G8, and/or G12 do not show this behavior. The observed changes in the pH-rate behavior are consistent with a mechanism in which N1 protonation-deprotonation events of guanine or 2,6-diaminopurine at positions 8 and 12 are essential for catalysis. Further support for the participation of G8 and G12 comes from photochemical cross-linking experiments, which show that G8 and G12 can stack upon the two substrate nucleobases at the reactive linkage, G(or U)1.1 and C17 (Heckman, J. E., Lambert, D., and Burke, J. M. (2005) Photocrosslinking detects a compact active structure of the hammerhead ribozyme, Biochemistry 44, 4148-4156). Together, these results support a model in which the hammerhead undergoes a transient conformational change into a catalytically active structure, in which stacking of G8 and G12 upon the nucleobases spanning the cleavage site provides an appropriate architecture for general acid-base catalysis. The hammerhead and hairpin ribozymes may share similarities in the organization of their active sites and their catalytic mechanism.

Alternative splicing is a process to differentially link exon regions in a single precursor mRNA to produce two or more different mature mRNAs, a strategy frequently used by higher eukaryotic cells to increase proteome diversity and/or enable additional post-transcriptional control of gene expression. This process can take place either co-transcriptionally or post-transcriptionally. When and where RNA splicing takes place in the cell represents a central question of cell biology; co-transcriptional splicing allows functional integration of transcription and RNA processing machineries, and could allow them to modulate one another, whereas post-transcriptional splicing could facilitate coupling RNA splicing with downstream events such as RNA export to create additional layers for regulated gene expression. This review focuses on recent advances in co- and post-transcriptional RNA splicing and proposes a new paradigm that some specific coupling events contribute to genome organization in higher eukaryotic cells.

SR proteins are well known to promote exon inclusion in regulated splicing through exonic splicing enhancers. SR proteins have also been reported to cause exon skipping, but little is known about the mechanism. We previously characterized SRSF1 (SF2/ASF)-dependent exon skipping of the CaMKII␦ gene during heart remodeling. By using mouse embryo fibroblasts derived from conditional SR protein knockout mice, we now show that SR protein-induced exon skipping depends on their prevalent actions on a flanking constitutive exon and requires collaboration of more than one SR protein. These findings, coupled with other established rules for SR proteins, provide a theoretical framework to understand the complex effect of SR protein-regulated splicing in mammalian cells. We further demonstrate that heart-specific CaMKII␦ splicing can be reconstituted in fibroblasts by downregulating SR proteins and upregulating a RBFOX protein and that SR protein overexpression impairs regulated CaMKII␦ splicing and neuronal differentiation in P19 cells, illustrating that SR protein-dependent exon skipping may constitute a key strategy for synergism with other splicing regulators in establishing tissue-specific alternative splicing critical for cell differentiation programs.The splicing machinery is largely conserved in eukaryotic cells. However, compared to budding yeast, where critical splicing signals are nearly invariant, higher eukaryotic cells rely on auxiliary factors to help define functional splice sites that are only loosely conserved. Most genes in higher eukaryotic cells also undergo alternative splicing, which is subject to regulation by a variety of RNA binding proteins (2). SR proteins are unique to higher eukaryotes and are among the best-characterized RNA binding proteins involved in both constitutive and regulated splicing (29,31,48). Intensive biochemical analysis in the past 2 decades has established that the RNA recognition motifs (RRMs) of SR proteins are responsible for sequence-specific binding to the pre-mRNA, whereas the RS domain appears to mediate both protein-protein and protein-RNA interactions during the splicing reaction (17, 39).Individual SR proteins exhibit distinct RNA binding specificities for various exonic splicing enhancers (ESEs), a second code in higher eukaryotic genomes that is critical for defining functional splice sites. In many cases, multiple SR proteins bind to several ESEs within the same exon, which is thought to provide redundant functions to ensure constitutive splicing against variation of SR proteins in different cell types and tissues. However, it has become abundantly clear that individual SR proteins are not functionally redundant in vivo (1, 4, 30). Because exons are short whereas introns are highly variable in length, functional splice sites in most mammalian genes are initially recognized by the exon definition mechanism, in which ESE-bound SR proteins promote U2AF recognition of the 3Ј splice site and U1 binding to the downstream 5Ј splice site across the exon (16). Initial exon...

Alternative pre-mRNA splicing has a major impact on cellular functions and development with the potential to fine-tune cellular localization, posttranslational modification, interaction properties, and expression levels of cognate proteins. The plasticity of regulation sets the stage for cells to adjust the relative levels of spliced mRNA isoforms in response to stress or stimulation. As part of an exon profiling analysis of mouse cortical neurons stimulated with high KCl to induce membrane depolarization, we detected a previously unrecognized exon (E24a) of the CASK gene, which encodes for a conserved peptide insertion in the guanylate kinase interaction domain. Comparative sequence analysis shows that E24a appeared selectively in mammalian CASK genes as part of a >3,000 base pair intron insertion. We demonstrate that a combination of a naturally defective 5′ splice site and negative regulation by several splicing factors, including SC35 (SRSF2) and ASF/SF2 (SRSF1), drives E24a skipping in most cell types. However, this negative regulation is countered with an observed increase in E24a inclusion after neuronal stimulation and NMDA receptor signaling. Taken together, E24a is typically a skipped exon, which awakens during neuronal stimulation with the potential to diversify the protein interaction properties of the CASK polypeptide.