Plant serine/arginine-rich proteins and their role in pre-mRNA splicing

Pre-messenger RNA (pre-mRNA) splicing, a process by which mature mRNAs are generated by excision of introns and ligation of exons, is an important step in the regulation of gene expression in all eukaryotes. Selection of alternative splice sites in a pre-mRNA generates multiple mRNAs from a single gene that encode structurally and functionally distinct proteins. Alternative splicing of pre-mRNAs contributes greatly to the proteomic complexity of plants and animals and increases the coding potential of a genome. However, the mechanisms that regulate constitutive and alternative splicing of pre-mRNA are not understood in plants. A serine/arginine-rich (SR) family of proteins is implicated in constitutive and alternative splicing of pre-mRNAs. Here I review recent progress in elucidating the roles of serine/arginine-rich proteins in pre-mRNA splicing.     

The 5′ UTR intron-mediated enhancement of constitutive splicing of the tobacco microsome ω-3 fatty acid desaturase gene

Several plant genes have their first intron in the 5′ untranslated region (5′ UTR), and such 5′ UTR introns often show several biological functions, including the intron-mediated enhancement of protein expression through an increase of mRNA level (IME), intron-dependent spatial expression, and intron-mediated enhancement of translation. Here, we show another function of the 5′ UTR intron, i.e., the 5′ UTR intron-mediated enhancement of constitutive splicing. The NtFAD3 gene, which encodes a tobacco microsome ω-3 fatty acid desaturase, has a 552 nucleotide-long 5′ UTR intron (intron 1), and the other seven introns are located in the coding sequence. The splicing of the 5′ half region of the NtFAD3 was studied through an in vivo splicing assay using Arabidopsis leaf explants. The low splicing efficiency of intron 2 was much improved when the assay construct harbored intron 1. Deletion of intron 1 and the replacement of intron 1 to the NtFAD3 intron 8 decreased the splicing efficiency of intron 2. The splicing enhancers were redundant and dispersed in the 5′ splice site-proximal, 284-nucleotides region of intron 1. In addition, the interaction among the cis-elements, i.e., the splicing enhancers in the intron 1 and exon 2, were necessary for the efficient splicing of intron 2. The 5′ UTR intron-mediated constitutive splicing was partially inhibited when an SR-like protein, SR45, was deficient. These results indicated a novel function of the 5′ UTR intron, namely an enhancement of the constitutive splicing.     

Regulation of alternative 3' splice site selection by constitutive splicing factors.

We have devised an in vitro splicing assay in which the mutually exclusive exons 2 and 3 of alpha-tropomyosin act as competing 3' splice sites for joining to exon 1. Splicing in normal HeLa cell nuclear extracts results in almost exclusive joining of exons 1 and 3. Splicing in decreased nuclear extract concentrations and decreased ionic strength results in increased 1-2 splicing. We have used this assay to determine the role of three constitutive pre-mRNA splicing factors on alternative 3' splice site selection. Polypyrimidine tract binding protein (PTB) was found to inhibit the splicing of introns containing a strong binding site for this factor. However, the inhibitory effect of PTB could be partially reversed if pre-mRNAs were preincubated with U2 auxiliary factor (U2AF) prior to splicing in PTB-supplemented extracts. For alpha-tropomyosin, regulation of splicing by PTB and U2AF primarily affected the joining of exons 1-3 with no dramatic increases in 1-2 splicing being detected. Preincubation of pre-mRNAs with SR proteins led to small increases in 1-2 splicing. However, if pre-mRNAs were preincubated with SR proteins followed by splicing in PTB-supplemented extracts, there was a nearly complete reversal of the normal 1-2 to 1-3 splicing ratios. Thus, multiple pairwise, and sometimes antagonizing, interactions between constitutive pre-mRNA splicing factors and the pre-mRNA can regulate 3' splice site selection.     

Association pattern mining of intron retention events in human based on hybrid learning machine

Alternative splicing is a main component of protein diversity, and aberrant splicing is known to be one of the main causes of genetic disorders such as cancer. Many statistical and computational approaches have identified several major factors that determine the splicing event, such as exon/intron length, splice site strength, and density of splicing enhancers or silencers. These factors may be correlated with one another and thus result in a specific type of splicing, but there has not been a systematic approach to extracting comprehensible association patterns. Here, we attempted to understand the decision making process of the learning machine on intron retention event. We adopted a hybrid learning machine approach using a random forest and association rule mining algorithm to determine the governing factors of intron retention events and their combined effect on decision-making processes. By quantifying all candidate features into five category values, we enhanced the understandability of generated rules. The interesting features found by the random forest algorithm are that only the adenine- and thymine-based triplets such as ATA, TTA, and ATT, but not the known intronic splicing enhancer GGG triplet is shown the significant features. The rules generated by the association rule mining algorithm also show that constitutive introns are generally characterized by high adenine- and thymine-based triplet frequency (level 3 and above), 3' and 5' splice site scores, exonic splicing silencer scores, and intron length, whereas retained introns are characterized by low-level counterpart scores.     

Use of minigene systems to dissect alternative splicing elements

Pre-mRNA splicing is an essential step for gene expression in higher eukaryotes. The splicing efficiency of individual exons is determined by multiple features involving gene architecture, a variety of cis-acting elements within the exons and flanking introns, and interactions with components of the basal splicing machinery (called the spliceosome) and auxiliary regulatory factors which transiently co-assemble with the spliceosome. Both alternative and constitutive exons are recognized by multiple weak protein:RNA interactions and different exons differ in the interactions which are determinative for exon usage. Alternative exons are often regulated according to cell-specific patterns and regulation is mediated by specific sets of cis-acting elements and trans-acting factors. Transient expression of minigenes is a commonly used in vivo assay to identify the intrinsic features of a gene that control exon usage, identify specific cis-acting elements that control usage of constitutive and alternative exons, identify cis-acting elements that control cell-specific usage of alternative exons, and once regulatory elements have been identified, to identify the trans-acting factors that bind to these elements and modulate splicing. This chapter describes approaches and strategies for using minigenes to define the cis-acting elements that determine splice site usage and to identify and characterize the trans-acting factors that bind to these elements and regulate alternative splicing.     

Analysis of the role of Caenorhabditis elegans GC-AG introns in regulated splicing

GC-AG introns represent 0.7% of total human pre-mRNA introns. To study the function of GC-AG introns in splicing regulation, 196 cDNA-confirmed GC-AG introns were identified in Caenorhabditis elegans. These represent 0.6% of the cDNA- confirmed intron data set for this organism. Eleven of these GC-AG introns are involved in alternative splicing. In a comparison of the genomic sequences of homologous genes between C.elegans and Caenorhabditis briggsae for 26 GC-AG introns, the C at the +2 position is conserved in only five of these introns. A system to experimentally test the function of GC-AG introns in alternative splicing was developed. Results from these experiments indicate that the conserved C at the +2 position of the tenth intron of the let-2 gene is essential for developmentally regulated alternative splicing. This C allows the splice donor to function as a very weak splice site that works in balance with an alternative GT splice donor. A weak GT splice donor can functionally replace the GC splice donor and allow for splicing regulation. These results indicate that while the majority of GC-AG introns appear to be constitutively spliced and have no evolutionary constraints to prevent them from being GT-AG introns, a subset of GC-AG introns is involved in alternative splicing and the C at the +2 position of these introns can have an important role in splicing regulation.     

Conserved sequence elements associated with exon skipping

One of the major forms of alternative splicing, which generates multiple mRNA isoforms differing in the precise combinations of their exon sequences, is exon skipping. While in constitutive splicing all exons are included, in the skipped pattern(s) one or more exons are skipped. The regulation of this process is still not well understood; so far, cis- regulatory elements (such as exonic splicing enhancers) were identified in individual cases. We therefore set to investigate the possibility that exon skipping is controlled by sequences in the adjacent introns. We employed a computer analysis on 54 sequences documented as undergoing exon skipping, and identified two motifs both in the upstream and downstream introns of the skipped exons. One motif is highly enriched in pyrimidines (mostly C residues), and the other motif is highly enriched in purines (mostly G residues). The two motifs differ from the known cis-elements present at the 5′ and 3′ splice site. Interestingly, the two motifs are complementary, and their relative positional order is conserved in the flanking introns. These suggest that base pairing interactions can underlie a mechanism that involves secondary structure to regulate exon skipping. Remarkably, the two motifs are conserved in mouse orthologous genes that undergo exon skipping.