The study presented here provides insights into the intricate mechanism that regulates SUS1 expression, a crucial factor linking transcription and mRNA export. We found that (i) SUS1 introns are required for its functionality, (ii) protein levels of Sus1 are controlled by SUS1 introns, (iii) SUS1 expression is regulated by splicing and NMD and (iv) intronic and exonic sequences participate in SUS1 regulation. The accompanying manuscript by Hossain et al. in this issue exposes the relevance of SUS1 expression as a model for understanding the role of core spliceosomal components in alternative splicing.
Discovery of the SUS1
gene in a genetic screening revealed an intriguing genomic organization (10
nt) consists of three exons (of 71, 140, and 77
nt) and two introns (80 and 70
nt). Notably, their size and position are widely conserved (Supplementary Table S2
). Sus1 plays important roles during transcription elongation and mRNA biogenesis, which likely explains its high degree of evolutionary conservation, from yeast to human. Why does this small gene, with key functions at different stages in gene expression, contains two introns in yeast, where most genes contain none? A provocative hypothesis is that this allows the SUS1
gene to be a sensor of these processes, acting in a yet unknown feedback control mechanism. We demonstrate here that splicing and decay of SUS1
transcripts regulate expression of Sus1 protein. This strategy is reminiscent of that of YRA1
, another factor involved in the coupling of mRNA export and transcription. Intriguingly, YRA1
shares with SUS1
having both an atypical intron and its expression regulated at the level of splicing, degradation and export of its transcripts, although likely by different mechanisms (22
). Moreover, mutations in SUS1
are synthetic lethal, and Sus1 and Yra1 interact physically. A possible scenario is that these factors are finely tuned to work together to sense or modulate correct mRNP biogenesis, as they are sensitive to alterations in several aspects of this pathway.
Our findings reveal that I1 is the major determinant of SUS1 splicing efficiency. Elimination of I1 or mutation of its BS leads to more efficient SUS1 splicing. These data are consistent with the observed weak splicing efficiency of I1, largely due to its non-consensus BS and imply that I1 and its BS are crucial for SUS1 expression. We also show that splicing of SUS1 largely depends on the BS recognition factor Mud2. However, we see that deletion of I1 has a stronger effect than mutation of the BS on mud2Δ cells. These results imply that other features of I1 are dependent on Mud2, likely the non-consensus 5′ splice site of I1 (GUAUGA). In agreement with this, Hossain et al., in the accompanying study have addressed the relevance of non-canonical splicing signals for I1 and I2, revealing that the 5′ splice site of I1 is in fact an important determinant of its processing.
We find an intriguing co-evolution between the presence of a non-consensus BS in I1 and the existence of a conserved long sequence at the 3′-end of the gene. In this context, transcriptome analyses showed that SUS1
transcripts retaining I1 carry a longer 3′UTR than the fully spliced RNAs (38
). We hypothesize that efficient splicing and proper 3′-end formation in SUS1
could be linked, as it has been shown for other transcripts (44
). Current work in our lab tries to address how important is this 3′-end sequence in SUS1
Intron 2 of SUS1 also appears to play an important role in splicing, despite its apparent lack of unusual features. We find that removing I2 can be detrimental for protein expression for LexA-SUS1 (A, lane 8), and intriguingly, the sole presence of I2 provokes a splicing block when placed into the TAF14 gene (A), which is consistent with the data from Hossain et al. addressing the splicing efficiency of I2. In addition, swapping SUS1 introns also affects Sus1 protein levels, strongly arguing for a co-dependence in splicing of both SUS1 introns. We also have found that splicing of SUS1 introns is also affected by exonic sequences, as evidenced by the low expression of TAF14-SUS1 chimerical constructs (). Remarkably this can be suppressed by replacing the TAF14 newly-created middle-exon with that of SUS1. Consistent with this, substituting SUS1 exon 2 by this TAF14 ‘exon 2’ reduces Cup1 expression in SUS1-CUP1 constructs. Thus our data suggest a positive role for exon 2 in SUS1 splicing. Work is currently under way to address whether exonic splicing enhancers are involved.
We have verified the biological relevance of SUS1
introns, by showing that a cDNA version of SUS1
is not able to fully complement sus1Δ
phenotypes in mRNA export and growth [; we also assess that this is not due to a lack of protein production from an intron-less construct (lanes 2 and 3 versus 4 and 5, B)]. Similar behavior was observed for YRA1
). Observations that SUS1
splicing is inefficient under optimal growth (A) suggest that SUS1
splicing could be regulated. Consistently, we find that the ratios between the different species of SUS1
RNAs are influenced by growth conditions. In fact, at higher temperatures in which SUS1
function is more critical, pre-mRNA forms retaining the I1 or both introns (I1 and I2) are more abundant (B). This change in the transcript ratios could account for the lack of fully complementation by SUS1-cDNA at 37°C (B and C). In this context a striking observation is the existence of a small protein containing part of Sus1, likely translated form a transcript containing I1 (). Western analyses are consistent with this peptide being produced, in agreement with the functional requirement of transcripts containing I1, suggesting a role of the I1 SUS1
transcripts. Our results are consistent with the notion that the small Sus1 peptide is not a proteolytic product of the full-length protein. First, it accumulates in mud2Δupf1Δ
cells, where full-length Sus1 production is reduced (); and second, the small Sus1 product is absent in cells bearing an intron-less SUS1
, where full-length Sus1 is enhanced (). An alternate explanation, invoking a selective degradation of full-length Sus1 when encoded by an intron-containing SUS1
gene, is difficult to conceptualize and not consistent with the abundance of the small peptide in cells bearing the SUS1
SL-9 BS mutant, which cannot be spliced and thus cannot encode full-length Sus1. Notably, the functional requirement of transcripts containing I1 at high temperature is suggestive of a biological role for this unprocessed SUS1
transcript. More work is needed to assign this functionality to the pre-mRNA, its product, or both.
Our results, together with those from the accompanying report of Hossain et al., provide compelling evidence indicating that expression of SUS1 hinges on a balance of several factors including limited splicing and degradation pathways, modulated by intronic and exonic sequences. The question now emerges as to how this complex strategy gives SUS1 its relevant role in multiple aspects of mRNP biogenesis.