Abstract
BackgroundSecondary structure interactions within introns have been shown to be essential for efficient splicing of several yeast genes. The nature of these base-pairing interactions and their effect on splicing efficiency were most extensively studied in ribosomal protein gene RPS17B (previously known as RP51B). It was determined that complementary pairing between two sequence segments located downstream of the 5' splice site and upstream of the branchpoint sequence promotes efficient splicing of the RPS17B pre-mRNA, presumably by shortening the branchpoint distance. However, no attempts were made to compute a shortened, 'structural' branchpoint distance and thus the functional relationship between this distance and the splicing efficiency remains unknown.ResultsIn this paper we use computational RNA secondary structure prediction to analyze the secondary structure of the RPS17B intron. We show that it is necessary to consider suboptimal structure predictions and to compute the structural branchpoint distances in order to explain previously published splicing efficiency results. Our study reveals that there is a tight correlation between this distance and splicing efficiency levels of intron mutants described in the literature. We experimentally test this correlation on additional RPS17B mutants and intron mutants within two other yeast genes.ConclusionThe proposed model of secondary structure requirements for efficient splicing is the first attempt to specify the functional relationship between pre-mRNA secondary structure and splicing. Our findings provide further insights into the role of pre-mRNA secondary structure in gene splicing in yeast and also offer basis for improvement of computational methods for splice site identification and gene-finding.
Highlights
Secondary structure interactions within introns have been shown to be essential for efficient splicing of several yeast genes
It was hypothesized that 5'L introns, for which the branchpoint distance is greater than 200 nt, can fold into secondary structures to optimize the positioning of the 5' splice site and branchpoint sequence to one that is optimal for spliceosome assembly [3]
The authors created 9 mutant introns within the RPS17B gene: 3mUB1 (3 nt mutation), 4mUB1 (4 nt), 5mUB1 (5 nt), 6mUB1 (6 nt) and 8mUB1 (8 nt), where mutations fall in the UB1 region; 3mDB1 (3 nt) and 5mDB1 (5 nt), where mutations fall in the DB1 region and are designed to restore the base-pairing disrupted by the mutations in the 3mUB1 and 5mUB1, respectively; and 3mUB1_3mDB1 and 5mUB1_5mDB1, which are double mutants
Summary
Secondary structure interactions within introns have been shown to be essential for efficient splicing of several yeast genes. It was hypothesized that 5'L introns, for which the branchpoint distance is greater than 200 nt, can fold into secondary structures to optimize the positioning of the 5' splice site and branchpoint sequence to one that is optimal for spliceosome assembly [3]. This hypothesis was confirmed for a limited number of yeast introns by comprehensive biological experiments that demonstrated that the existence of such secondary structure elements is essential for splicing efficiency [6,7,8,9,10,11]. In mammalian cells, folding of long intron sequences is facilitated by protein binding and interactions, which presumably shortens the long distance between essential splicing sequences [13]
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