Regulated meiotic splicing in budding yeast is an elegant example of a post-transcriptional ‘off–on’ switch linked to a developmental program. Whereas only ~5% of yeast genes contain introns, there is an evident enrichment for introns among the genes encoding meiosis-specific proteins, and a tendency for meiotic introns to bear splicing signals that deviate from the norm (31
). It has been suggested that upregulation of splicing during meiosis—via programmed transition from the splicing ‘off’ state during vegetative growth—provides added protection against the untimely production of meiotic proteins that could be deleterious to vegetative cells (11
). Prior studies had characterized a single meiotic splicing regulon controlled by Mer1 and Nam8 and embracing three pre-mRNA targets. The regulatory inputs (if any) to the splicing of the other known meiosis-specific pre-mRNAs are a virtual tabula rasa
). Here, by surveying the full catalogue of meiosis-specific spliced pre-mRNAs for their dependence on Nam8, we’ve expanded the scope of the Mer1/Nam8 regulon to embrace SPO22
and illuminated a novel Mer1-independent role for Nam8 in splicing of PCH2
pre-mRNA. The two flavors of Nam8-dependent splicing differ fundamentally. The Mer1/Nam8-regulated meiotic transcripts share two properties: (i) they have an intronic enhancer sequence, to which Mer1 is thought to bind via its KH domain and (ii) they have non-consensus 5′ splice sites. By contrast, the PCH2
transcript lacks a Mer1 intronic enhancer and contains a perfect 5′ splice site.
We focused here on dissecting the contributions of the Mer1 enhancer sequence, Mer1 KH domain, and other SPO22 intronic signals to splicing of SPO22 pre-mRNA, the newly identified Nam8 target. Whereas SPO22 has multiple potentially enfeebling non-consensus intronic signals (5′SS, BP and 3′SS), we find that the atypical 5′SS is the decisive factor in Nam8/Mer1 dependence, insofar as a single nucleotide change in the SPO22 intron that restores a consensus 5′SS overrides the requirements for Mer1 and Nam8. The same scenario applies to the MER3 intron.
Our studies of PCH2
splicing implicate the non-consensus 5′-CACUAAC branchpoint and the exceptionally long 5′ exon as separable negative influences on PCH2
splicing in wild-type cells and as concerted determinants of Nam8-dependence. The finding that Nam8-dependence is portable with the PCH2
intron, when inserted into the HIS3
reporter, certifies the importance of the intron and its deviant branchpoint as decisive elements per se
in the Nam8 requirement. The action of Nam8 in countering a suboptimal PCH2
branchpoint in the context of a perfect 5′ splice site differs from the case of Mer1/Nam8-dependent splicing, where the intron-bound Mer1 facilitates recruitment of the Nam8-containing U1 snRNP to a suboptimal 5′ splice site (14
) (A). Current models invoke bridging contacts between U1 snRNP components Nam8, Snu56 and Snu71 with the enhancer-bound Mer1 in mediating U1 recruitment to the deviant 5′ splice site (22
) (A). We speculate that Nam8 facilitates PCH2
splicing via macromolecular interactions that recruit the U2 snRNP to the deviant branchpoint (B). The participants in this putative interaction network remain to be identified. Toward that end, the Nam8-responsive HIS3-[PCH2]
reporter that we developed here may prove useful in forward genetic screening for yeast mutants that either lose their His prototrophy in NAM8
cells (as candidate cofactors for Nam8-dependent splicing) or mutants that acquire His prototrophy in nam8
cells (as candidate suppressors of Nam8-dependency).
Figure 11. Two modes of Nam8-dependent splicing. (A) The Mer1/Nam8 co-dependent splicing regulon embraces four meiotic pre-mRNAs with weak non-consensus 5′ splice sites (5′-SS*). Efficient splicing is achieved when Mer1, bound to an intronic enhancer (more ...)
The present study illuminates a first set of instructive structure–function relations for the yeast Nam8 protein. As depicted in and , Nam8 consists of three RRM domains flanked by N-terminal ‘leader’ and C-terminal ‘trailer’ segments. Whereas the distal segment of the trailer domain (aa 455–523) is unnecessary for any Nam8 activity surveyed, our results demonstrate an essential contribution by the proximal part of the Nam8 C-terminal domain (aa 401–454) in all aspects of Nam8 function. One or more of the RRMs are plausible candidates for the imputed intron RNA-binding properties of Nam8. Here, we showed that the N leader and RRM1 are dispensable for Nam8-dependent splicing of meiotic pre-mRNA targets and for Nam8 function in vegetative yeast growth in a variety of synthetic genetic backgrounds. Thus, RRM1 is not implicated in Nam8′s principal contacts to RNA or other components of the splicing apparatus.
Deleting RRM2 along with RRM1 resulted in: (i) disabled Nam8-dependent splicing of MER2
pre-mRNAs; (ii) variable effects, ranging from loss of activity to no impact, on Nam8-dependent growth, according to the genetic background being assayed. Because it is possible that RRM1 and RRM2 are functionally redundant, we made an independent test of RRM2 function by targeting alanine substitutions to the putative RNA-binding surface of RRM2 in the context of an otherwise active Nam8 protein that contains all three RRMs. The decrements in vegetative growth in the tgs1
background and in splicing of MER2
pre-mRNA implicated RRM2 as a direct participant in Nam8 activity. Nakagawa and Ogawa (21
) reported previously that a single mutation, Leu170Pro, in the RNP2 motif (IFVGDL170
) of Nam8′s RRM2 domain resulted in a severe defect in meiotic recombination, comparable to that of a nam8
null. However, we deem this mutational effect as poorly instructive with regard to RRM function, because reference to the equivalent VFVGDL motif in the TIA-1 RRM2 crystal structure (B) shows us that the Leu170 side chain is not projecting outward onto the putative RNA-binding surface, but rather points into the hydrophobic core of the RRM (data not shown). Thus, a proline change at this position would be more likely to impact the folding of the RRM2 module than to specifically affect an RNA-binding site.
Extending the N-terminal deletions to the proximal margin of RRM3 (aa 304) abolished or severely weakened all of the Nam8 functions tested. As mentioned above, we attempted to isolate RRM3′s contributions by making alanine changes in the predicted RRM3 RNA-binding surface. The severe defects in vegetative growth in the tgs1
background and in splicing of MER2
pre-mRNA caused by the RRM3 alanine clusters implicated RRM3, too, as important for Nam8 activity. Although the results are consistent with an RNA-binding role for either RRM2 or RRM3, it is plausible that one (or both) of the Nam8 RRMs mediates important protein–protein interactions, rather than RNA binding, as has been described for other RRM domains (50
It is interesting to note that the least demanding genetic function of Nam8 is seen in the mud1
background, where Nam8-(291–454)—which lacks RRMs 1 and 2—suffices for normal growth. Mud1, like Nam8, is an intrinsic protein component of the yeast U1 snRNP. We presume that the synthetic growth phenotype of the nam8Δ mud1Δ
double-mutant reflects a gross defect in U1 snRNP structure and function, in which case its full complementation by Nam8-(291–454) implies that this minimized version of Nam8 is assimilated into the U1 snRNP lacking Mud1 and renders it active. That Nam8-(291–454) is not adept at (i) splicing Nam8 meiotic targets with suboptimal introns; (ii) complementing yeast synthetic lethal interactions with splicing factors that are not U1 snRNP components (Lea1, Mud2, Tgs1); or (iii) inhibiting vegetative growth when overexpressed suggests that upstream domains (i.e. RRM2 and the interdomain linker) mediate the invoked interactions of Nam8 with non-U1 splicing factors or the pre-mRNA.