Position-Dependent Repression and Promotion of DQB1 Intron 3 Splicing by GGGG Motifs

Abstract

Alternative splicing of HLA-DQB1 exon 4 is allele-dependent and results in variable expression of soluble DQβ. We have recently shown that differential inclusion of this exon in mature transcripts is largely due to intron 3 variants in the branch point sequence (BPS) and polypyrimidine tract. To identify additional regulatory cis-elements that contribute to haplotype-specific splicing of DQB1, we systematically examined the effect of guanosine (G) repeats on intron 3 removal. We found that the GGG or GGGG repeats generally improved splicing of DQB1 intron 3, except for those that were adjacent to the 5′ splice site where they had the opposite effect. The most prominent splicing enhancement was conferred by GGGG motifs arranged in tandem upstream of the BPS. Replacement of a G-rich segment just 5′ of the BPS with a series of random sequences markedly repressed splicing, whereas substitutions of a segment further upstream that lacked the G-rich elements and had the same size did not result in comparable splicing inhibition. Systematic mutagenesis of both suprabranch guanosine quadruplets (G4) revealed a key role of central G residues in splicing enhancement, whereas cytosines in these positions had the most prominent repressive effects. Together, these results show a significant role of tandem G4NG4 structures in splicing of both complete and truncated DQB1 intron 3, support position dependency of G repeats in splicing promotion and inhibition, and identify positively and negatively acting sequences that contribute to the haplotype-specific DQB1 expression.

Negative and Positive mRNA Splicing Elements Act Competitively To Regulate Human Immunodeficiency Virus Type 1 Vif Gene Expression

“…In contrast, mutations of the downstream 5Јss-proximal GGGG motif result in increased splicing, suggesting that in this case, the motif acts as a splicing silencer. It was found in this study that point mutations within the second and third G residues had the most effect on splicing enhancement, but similar mutations in the 5Јss-proximal GGGG silencer were not tested (15). In the case of the brain region-specific C1 cassette exon of the GRIN1 transcript, mutation of the central two G residues of the 5Ј-proximal GGGG motif was shown to inactivate the silencer activity, but the effect of mutating only the first or fourth G residues of the GGGG motif was not tested (9).…”

“…Previous studies have shown that a GGGG motif proximal to the 5Јss of the brain region-specific C1 cassette exon of the N-methyl-Daspartic acid (NMDA)-type glutamate receptor NR1 subunit (GRIN1) transcript acts to silence inclusion of the C1 exon (9). Several GGGG motifs in the intron sequences upstream and downstream of alternative exon 4 of the HLA-DQB1 gene play a role in the differential inclusion of exon 3 into mRNA (15). In the HIV-1 genome, there is a GGGG motif which overlaps the last base of the U1 snRNP binding site and is just proximal to the 5Јss D2 sequence.…”

Modulating role of RNA structure in alternative splicing of a critical exon in the spinal muscular atrophy genes

“…It has also been shown that intronic G triplets located immediately downstream of 5′ ss directly interact with U1 snRNA facilitating splice site recognition and functioning as ISE (87). On the other hand, G quadruplets adjacent to the 5′ ss have been shown to promote silencing of a brain-region-specific exon 19 of the GRIN1 gene (65) and inhibit the splicing of intron 3 in DQB1 reporter construct (88). Interestingly, the GGG/gu sequence similar to the one created by 54G was found to be inhibitory for NF1 exon 3 inclusion (89).…”

Ex vivo splicing assays of mutations at noncanonical positions of splice sites in USHER genes

“…These sequences have the ability to form highly stable quadruplex structures (G-quartet) and are involved in many different biological processes, including alternative splicing, particularly when they are associated with 5 0 ss with intermediate scores [Xiao et al, 2009]. Several hnRNPs proteins, including hnRNP H but also hnRNP A1, A2, and F have also been shown to bind G-rich RNA sequences and to regulate splicing both positively and negatively in a position dependent manner [Buratti et al, 2004;Kralovicova and Vorechovsky, 2006]. Whether these sequences play a role in the context of the USH1C c.121016T4G and USH2A c.14582139C4T mutations would require further investigations.…”