U1 snRNP-dependent function of TIAR in the regulation of alternative RNA processing of the human calcitonin/CGRP pre-mRNA.

Alternative RNA processing is an important means of increasing proteomic complexity in eukaryotic organisms. Through this process, the majority of human pre-mRNAs generate more than one mRNA molecule, which often leads to production of multiple polypeptides from a single gene (5, 20, 42). Tissue-specific alternative RNA processing plays an important role in regulating gene expression. The human calcitonin/calcitonin gene-related peptide (CGRP) gene is a good example of such regulation (1, 46). This gene is predominantly expressed in two tissues, thyroid C cells and a subset of neurons. However, the pre-mRNA for calcitonin/CGRP is differentially processed in these two tissues, leading to production of calcitonin, a calcium-regulating peptide hormone, in thyroid C cells and CGRP, a neurotransmitter, in neuronal cells (Fig. ​(Fig.1A1A). FIG. 1. The U tract sequence in the calcitonin/CGRP gene intron 4 enhancer element is required for exon 4 inclusion. (A) Schematic diagram of the calcitonin/CGRP gene and its alternative RNA processing in thyroid and neuronal cells. A, polyadenylation signal. ... Alternative RNA processing is subject to complex control, often involving multiple sequence elements and protein factors. Studies to date have identified a large number of cis-acting regulatory elements located in both exons and introns. In contrast, our understanding of what trans-acting factors regulate alternative RNA processing and how they do so is very limited. Only a small number of factors have been identified and characterized (4, 37). Studies on alternative processing factors have indicated that they are often RNA binding proteins capable of binding to specific cis-acting RNA elements and that they affect RNA processing choice through interactions with the basal splicing or polyadenylation machinery (4, 37). It has been estimated that the human genome may encode as many as 2,560 RNA binding proteins (25). There are at least 300 human genes that encode one or more RNA recognition motifs (RRM) (28). We are only beginning to uncover the functions of these proteins. We use the human calcitonin/CGRP gene as a model to study the mechanism controlling tissue-specific alternative RNA processing. Similar to that of many other alternatively processed pre-mRNAs, alternative processing of calcitonin/CGRP pre-mRNA undergoes complex control involving multiple sequence elements and protein factors (32). One important sequence element is located in the intron downstream of exon 4 (Fig. ​(Fig.1B).1B). This element is highly conserved between human, mouse, and rat and is required for exon 4 inclusion (36). One role of the intron element is to promote polyadenylation at the end of exon 4, thereby enhancing exon 4 inclusion (33, 35). Two interesting features make this enhancer element an intriguing target to study. First, the intron element reflects the complex nature of RNA elements regulating alternative RNA processing by containing multiple sequence motifs. In particular, both positive and negative motifs that regulate exon 4 inclusion exist within the element. Second, some of the motifs bear a striking resemblance to the consensus sequences of splicing signals yet are not recognized as authentic splice sites. Specifically, the major positive motifs include a pseudo 5′ splice site preceded by a pyrimidine-rich sequence and followed by a U tract (Fig. ​(Fig.1C).1C). Mutation of any of the three motifs drastically reduces exon 4 inclusion in cell types normally expressing RNA containing this exon (36). The major negative sequence motif is located 23 nucleotides (nt) upstream of these motifs and contains an additional 5′ splice site sequence. In contrast to that of the downstream 5′ splice site sequence, mutation of this upstream 5′ splice site sequence increased exon 4 inclusion significantly (36). It has been subsequently demonstrated that the serine- and arginine-rich protein SRp20 and the U1 small nuclear ribonucleoprotein particle (U1 snRNP) interact with the pyrimidine-rich sequence and the downstream pseudo 5′ splice site, respectively, thereby promoting polyadenylation of exon 4 (33, 35). The polypyrimidine tract binding protein (PTB) also interacts with the pyrimidine sequence and plays a positive role in regulating exon 4 inclusion (34), but the precise role of PTB in this regulation remains unclear. Although we have learned a great deal about the regulation of calcitonin/CGRP alternative RNA processing, it is clear that many questions remain to be answered. This report addresses two of them: what factors interact with the two pseudo 5′ splice sites and what factors interact with the U tract sequence following the downstream site. To fully understand the activity of the enhancer element, we set out to determine why the two 5′ splice sites function differently by characterizing the factors that interact with each. In this report, we demonstrate the interactions of two novel factors with the sequences at or close to the downstream pseudo 5′ splice site. In particular, we found that TIAR, a recently characterized splicing regulator (30), specifically interacts with the U tract sequence that follows the downstream pseudo 5′ splice site in the intron enhancer element. In addition, psoralen cross-linking assays indicate an expected interaction between U1 snRNA and both of the pseudo 5′ splice sites as well as an unexpected interaction between U6 snRNA and the positively acting downstream site. The U6 snRNP interaction depends on the downstream pseudo 5′ splice site sequence but not on the upstream 5′ splice site. We provide evidence to support the idea that the TIAR protein plays an important role in regulating exon 4 inclusion. Furthermore, our studies uncovered a novel function of TIAR protein as an alternative splicing regulator. Specifically, TIAR appears to have little effect on the interaction between U1 snRNP and the pseudo 5′ splice site in the intron enhancer. Instead, TIAR promotes an interaction between U6 snRNP and the pseudo 5′ splice site. Moreover, binding of TIAR protein and U6 snRNP to their RNA targets showed a synergistic relationship. These studies have demonstrated the versatile activities of TIAR protein as a regulator of alternative RNA processing. Additionally, they strongly suggest a novel role of U6 snRNP in regulated alternative RNA processing. Finally, involvement of TIAR and U6 snRNP in the intron enhancer function predicts a different and novel role for this enhancer element in addition to enhancement of polyadenylation.