An intronic RNA structure modulates expression of the mRNA biogenesis factor Sus1

Abstract

Sus1 is a conserved protein involved in chromatin remodeling and mRNA biogenesis. Unlike most yeast genes, the SUS1 premRNA of Saccharomyces cerevisiae contains two introns and is alternatively spliced, retaining one or both introns in response to changes in environmental conditions. SUS1 splicing may allow the cell to control Sus1 expression, but the mechanisms that regulate this process remain unknown. Using in silico analyses together with NMR spectroscopy, gel electrophoresis, and UV thermal denaturation experiments, we show that the downstream intron (I2) of SUS1 forms a weakly stable, 37-nucleotide stem-loop structure containing the branch site near its apical loop and the 3 ′ splice site after the stem terminus. A cellular assay revealed that two of four mutants containing altered I2 structures had significantly impaired SUS1 expression. Semiquantitative RT-PCR experiments indicated that all mutants accumulated unspliced SUS1 pre-mRNA and/or induced distorted levels of fully spliced mRNA relative to wild type. Concomitantly, Sus1 cellular functions in histone H2B deubiquitination and mRNA export were affected in I2 hairpin mutants that inhibited splicing. This work demonstrates that I2 structure is relevant for SUS1 expression, and that this effect is likely exerted through modulation of splicing.

Introns regulate the production of ribosomal proteins by modulating splicing of duplicated ribosomal protein genes

“…However, the reason behind this difference in the paralogs splicing efficiency is unclear. It was previously shown that intronic secondary structure might regulate splicing in yeast ( 27 – 29 ). Therefore, we compared the secondary structure of the RPS9A and RPS9B introns with the goal of identifying possible paralog-specific structural elements that might explain the difference in splicing efficiency.…”

An exon three-way junction structure modulates splicing and degradation of the SUS1 yeast pre-mRNA

“…We previously detected a reduction in SUS1 expression when cells are incubated at higher temperatures (for instance 20 min at 42 ºC), accompanied by accumulation of unspliced SUS1 transcripts and decrement of fully spliced mRNA [20]. Moreover, we recently showed that SUS1 I2 structural mutants affected the ratio of the different species in these conditions [22]. We thus tested the effect of destabilizing and restoring the P1 stem at 42 ºC.…”

“…On the other hand, the second intron (I2) is efficiently spliced and forms a weakly stable stem-loop structure that increases the accessibility of the BS and 3'SS nucleotides (nt). Changes in this I2 hairpin structure were found to alter the patterns of Sus1 expression as well as SUS1 splicing, giving rise to I1 retention and skipping of the second exon (E2) [22] (Figure 1). Altogether, these findings suggest that the functions of Sus1 in mRNA biogenesis are modulated via splicing regulation.…”

IDENTIFICATION OF RNA STRUCTURES MODULATING THE EXPRESSION OF THE mRNA BIOGENESIS FACTOR SUS1

“…It is worth noting in this regard that this phenotype was observed under stress conditions, a situation in which the ratio of the different SUS1 transcripts is more regulated (Cuenca-Bono, García-Molinero et al 2011;AbuQattam, Gallego et al 2016). These results demonstrate that similarly to intron 2 RNA structure , intrinsic features of E2…”

“…cerevisiae genes are intronless, the SUS1 gene consists of three exons (of 71, 140 and 77 nt) and two introns (of 80 and 70 nt), and its splicing and expression levels are regulated by different mechanisms [1], [52]. [53], [54].…”