A regulator of Dscam mutually exclusive splicing fidelity

Abstract

The Down syndrome cell adhesion molecule (Dscam) gene has essential roles in neural wiring and pathogen recognition in Drosophila melanogaster. Dscam encodes 38,016 distinct isoforms via extensive alternative splicing. The 95 alternative exons in Dscam are organized into clusters that are spliced in a mutually exclusive manner. The exon 6 cluster contains 48 variable exons and uses a complex system of competing RNA structures to ensure that only one variable exon is included. Here we show that the heterogeneous nuclear ribonucleoprotein hrp36 acts specifically within, and throughout, the exon 6 cluster to prevent the inclusion of multiple exons. Moreover, hrp36 prevents serine/arginine-rich proteins from promoting the ectopic inclusion of multiple exon 6 variants. Thus, the fidelity of mutually exclusive splicing in the exon 6 cluster is governed by an intricate combination of alternative RNA structures and a globally acting splicing repressor.Pre-mRNA splicing is frequently used to regulate important biological processes. It is currently estimated that between 60% and 75% of human genes encode alternatively spliced pre-mRNAs 1,2 . Though most of these genes encode 2-3 different isoforms, there are several examples of genes that can generate much larger repertoires of mRNAs. For example, the human KCNMA1 gene can generate ~500 different isoforms, and the three NRXN genes together produce over 2,000 isoforms 3,4 . However, by far the most notable example is the D. melanogaster Dscam gene, which can generate 38,016 isoforms 5 .Correspondence should be addressed to B.R.G. (graveley@neuron.uchc.edu). (Fig. 1a). These variable exons are organized in clusters; the exon 4, 6, 9 and 17 clusters contain 12, 48, 33 and 2 variable exons, respectively. Importantly, the exons within each cluster are spliced in a mutually exclusive manner-only one exon from each cluster is included in the mRNA. The exon 4, 6 and 9 clusters encode alternative versions of extracellular immunoglobulin repeats, whereas the exon 17 cluster encodes two different transmembrane domains, such that each of the 38,016 isoforms has the same overall domain structure.From a mechanistic view one of the most puzzling aspects of Dscam is how the splicing of the large clusters of exons occurs in a mutually exclusive manner. Thousands of eukaryotic genes contain regions that have two mutually exclusive exons, and a variety of mechanisms are known that ensure that only one exon is included in these mRNAs. These mechanisms involve steric hindrance, the use of both the major and minor spliceosomes, and the nonsense-mediated decay pathway 15 . However, none of these mechanisms explain how the variable clusters in Dscam can be spliced in a mutually exclusive manner.Insight into the mechanism by which Dscam splicing occurs in a mutually exclusive manner was obtained from comparative genomics 16,17 . The exon 6 cluster contains two classes of conserved elements: the docking site, which is located in the intron downstream of constitutive exon 5, and the sele...

Massive expansions of Dscam splicing diversity via staggered homologous recombination during arthropod evolution

“…While the mechanism of mutually exclusive splicing has been studied in both the exon 4 (Kreahling and Graveley 2005) and exon 6 clusters (Graveley 2005;Anastassiou et al 2006;Olson et al 2007), the mechanism is only well understood for the exon 6 cluster. Mutually exclusive splicing within the exon 6 cluster involves the formation of competing RNA secondary structures between selector sequences located upstream of each variable exon and a single docking site located between exon 5 and the first exon 6 variant (Graveley 2005;Anastassiou et al 2006).…”

Conservation and regulation of alternative splicing by dynamic inter- and intra-intron base pairings in Lepidoptera 14-3-3z pre-mRNAs

“…6 Several trans-acting factors and multiple cis-elements have been demonstrated to regulate the mutually exclusive exons in cultured insect and mammalian cells. [7][8][9][10][11][12][13][14][15][16] Several mechanisms have been proposed to direct mutually exclusive splicing, including steric interference, 17 spliceosome incompatibility, 18 nonsense-mediated decay, 6 and competing RNA secondary structures. [19][20][21][22] However, the underlying regulatory mechanisms in large part remain poorly understood.…”

“…For example, the process of Dscam and 14-3-3ζ variable exon choice in D. melanogaster relies on a combination of alternative RNA structure and repressors, which act in concert to activate a variable exon. 14,22 First, the latter arrangement would be advantageous when considering the evolutionary expansion from the two-duplicate-exon cluster to a three-duplicate-exon or a multiple-exon cluster. 22 Some complex alternative splicing has been achieved by expansion of the exon cluster through nonallelic homologous recombination.…”

Emerging Roles for hnRNPs in post-transcriptional regulation: what can we learn from flies?

“…In particular, work conducted in different groups demonstrated that changes in the abundance of hnRNPs could modulate alternative splicing (Borah et al 2009;Nichols et al 2000;Olson et al 2007;Nilsen and Graveley 2010). Though the molecular mechanism underlying this effect is not clearly understood, Valcarcel and colleagues recently provided evidence that the alternative splicing forms of the hnRNP Squid can contribute to sexspecific splicing during sex determination events (Hartmann et al 2011).…”

“…Grk, osk and nos and mRNAs are in orange, green and violet lines, respectively. stages to regulate the translation of neuronal mRNAs (Piper and Holt 2004;Martin 2005;Kalifa et al 2006;Olson et al 2007). Moreover, the hnRNP Hrb87F, in addition to its role in omega speckle formation as well as normal development and stress tolerance , is also widely involved in neuronal development.…”