During mRNP biogenesis, a network of cross-stimulatory connections and physical interdependencies is crucial for the formation of an export-competent mRNP (reviewed in references 12
). Cotranscriptional recruitment of splicing factors constitutes an integral part of mRNP formation. In yeast, the ordered association of spliceosomal components during transcription has been shown to facilitate efficient cotranscriptional mRNA processing (19
). In the current study, we define a novel role for protein arginine methylation in the cotranscriptional recruitment of pre-mRNA splicing factors. We have identified Snp1, a component of the canonical U1 snRNP (which participates in the early steps of spliceosomal complex assembly during transcription [60
]), as a novel substrate of Hmt1 in vitro
. Using ChIP-chip, we uncovered a role for protein arginine methylation in modulating the recruitment of splicing factors to ICGs. A number of these data were validated by directed ChIP experiments. Changes in the cotranscriptional recruitment of splicing factors due to the loss of arginine methylation affects regulated splicing. We show that the absence of arginine methylation leads to an aberrant interaction between Snp1 and the SR-like protein Npl3. Overall, our data demonstrate that protein arginine methylation plays a critical role in defining proper cotranscriptional recruitment of pre-mRNA splicing factors.
To date, studies characterizing the recruitment of pre-mRNA splicing factors on a genome-wide scale have focused on measuring changes in the absolute number or percentage of ICGs bound by a splicing factor (32
). This kind of measurement does not reveal how the distribution of bound ICGs changes in the context of a given condition or mutation. It is possible, for example, that a splicing factor is bound to the identical number and complement of ICGs under different conditions but that the ICGs bound in one case occupy the highly bound portion of the total bound population and in the other occupy the least-bound portion. Thus, the IDI serves as a novel method to assay the relative changes in ICG association when comparing two ChIP-chip profiles. In the study here, it reveals that the loss of Hmt1 or of its activity perturbs the relative degree of occupancy of ICGs for all U snRNPs examined, with the exception of U1 snRNP and its associated proteins.
Since the dynamics of U1 and U2 snRNP recruitment are intimately linked (19
), it is likely that a change in the recruitment dynamics of the U1 snRNP would produce a downstream effect on the recruitment of U2 and other U snRNPs. This could be achieved by perturbing the formation of the commitment complex. Our ChIP-chip data are consistent with such an effect, as they demonstrate that recruitment of the U2 snRNP and the U4/U5/U6 snRNP, as well as of their associated proteins, is reduced in Hmt1 mutants relative to that in wild-type cells. Since the maximal U1 snRNP recruitment occurs near the 5′ ss (64
), it is likely that the loss of arginine methylation in Hmt1 mutants results in increased retention of the U1 snRNP and its associated proteins at ICGs. This may be reflected by the observed decrease in binding of U1 snRNP and its associated proteins at non-ICGs, since fewer U1 snRNPs would be available to “scan” the non-ICGs as they are effectively “titrated away” as a consequence of longer ICG retention. Disruption of this in vivo
recruitment dynamic could theoretically prevent recognition of the other components of the spliceosomal complex (such as proteins from the U2 snRNP and the U4/U5/U6 tri-snRNP) and thus their efficient recruitment to genomic targets. This could give rise to a scenario in which formation of the commitment complex is compromised. Indeed, our ChIP-chip data—according to which the U2 snRNP, the U4/U5/U6 tri-snRNP, and their associated proteins all exhibited lower relative affinities for ICGs in Hmt1 mutants—support such a scenario.
Our genome-wide assays led to the identification of a meiosis-related gene whose transcripts display enhanced splicing during vegetative growth in Hmt1 mutants. The observation that the loss of arginine methylation leads to aberrant recruitment of splicing factors and misregulated splicing of these targets supports our previous study, in which we showed that differential spliceosome recruitment predicts regulated splicing in precisely the same population of genes, i.e., meiosis-related ICGs (47
). The relatively small increases in the pre-mRNA splicing of HOP2
may be due to the nuclear mRNA surveillance machinery monitoring sites of regulated splicing, since increased levels of unspliced meiotic mRNAs have been observed in Δrrp6
cells, which lack a subunit of the nuclear exosome (47
). Testing a mutant in which both HMT1
are deleted may reveal more dramatic changes. Notably, Δhmt1
cells display decreased sporulation frequency (16
). Whether this abnormality results from altered splicing of meiosis-related transcripts remains to be investigated. Nevertheless, our present study has established arginine methylation as a requirement for regulated recruitment of the spliceosome.
Arginine methylation has been demonstrated to affect the function of protein substrates by modulating their interactions with other proteins (5
). For example, the interaction of mammalian PRMT1 and transcription factor Ying Yang 1 (YY1) is necessary for the recruitment of histone H4-specific methyltransferase activity (54
). Hmt1 has been demonstrated to control the biochemical association between mRNA export factor Npl3 and transcriptional elongation factor Tho2, as well as the self-association of Npl3 (71
). While the predominant role of Npl3 is in the export of mRNAs (29
), it was recently revealed that Npl3 also promotes pre-mRNA splicing, as it is required for the cotranscriptional recruitment of early splicing factors such as the U1 snRNP protein Prp40 and the U2 snRNP protein Lea1 (33
). Furthermore, Npl3 is a yeast SR-like protein (15
), and SR proteins in mammalian cells are known to help stabilize the U1 snRNP during the spliceosome assembly (69
). These facts suggested that the loss of Hmt1 or its activity would impact how Npl3 interacts with the early splicing factors and that this interaction might account for the decreased recruitment of U2 and U4/U5/U6 tri-snRNP proteins in our ChIP-chip experiment. In support of this hypothesis, we observed an aberrant biochemical association between Npl3 and Snp1 in both the Δhmt1
mutants (Fig. ). Since Hmt1 also methylates Npl3, it is possible that arginine methylation regulates the cotranscriptional recruitment of splicing factors by altering Npl3 recruitment. However, our previous analysis shows that the loss of Hmt1 does not alter the recruitment of Npl3 to either ICGs or non-ICGs (see Fig. S4 in the supplemental material). Rather, the dynamics of biochemical association between Npl3 and Snp1 are most likely to control how the rest of the splicing factors are recruited in the context of mRNP biogenesis. New studies, using methylation-specific mutants of both Npl3 (45
) and Snp1, are under way to address the influence of methylation on the ability of each factor to promote this association.
A recent study demonstrated that the chromatin-modifying activity of the histone acetyltransferase Gcn5 is functionally linked to the cotranscriptional recruitment of pre-mRNA splicing factors (21
). Specifically, Gcn5 was found to acetylate the ICG-bound histone H3 (21
). Notably, a screen used to identify protein-protein interactions that are triggered by posttranslational modifications identified Hmt1 as a binding partner for acetylated histones (22
). Hmt1 binds both acetylated histones H3 and H4 but methylates only H4 (34
). Given that Hmt1 methylates histone H4 at position 3 (H4R3) in a chromatin-specific context (72
) and that Hmt1 loss does not abolish bulk changes in H4R3 methylation (38
), it would be interesting to determine whether Hmt1 plays a role in the status of H4R3 methylation within ICG-bound histones. Such a study would determine whether the chromatin-modifying activity of Hmt1 is linked to its role in optimizing the cotranscriptional recruitment of splicing factors.
Overall, our data support a model in which Hmt1-catalyzed arginine methylation controls the cotranscriptional recruitment of splicing factors by promoting proper Npl3-Snp1 interaction. Npl3 is cotranscriptionally recruited during the early stages of transcription as part of mRNP biogenesis. As a yeast SR-like protein, it may stabilize the U1 snRNP, much as SR family proteins do in mammalian cells. During the early phase of the transcription process, the Snp1-containing U1 snRNP samples transcribed genes to identify any introns. The detection of an intron leads to the formation of a commitment complex, followed by subsequent recruitment of the rest of the spliceosome. Given that mRNP assembly involves a multitude of associations and dissociations of its components, aberrant interactions between Npl3 and Snp1 in the Hmt1 mutants may cause the U1 snRNP proteins to be retained at their genomic targets for longer times than optimal. This disruption in the dynamics of U1 snRNP recruitment, in turn, would likely affect the formation of the commitment complex, judging by the ChIP-chip data for Prp11 and Mud2. Alternatively, it is possible that a change in the dynamics of U1 snRNP recruitment due to Hmt1 activity results in a rearrangement within the spliceosome that subsequently prevents proper recruitment of the U2 and U4/U5/U6 tri-snRNP components. In summary, our study establishes a novel and key regulatory role for protein arginine methylation in controlling the dynamics of cotranscriptional recruitment of pre-mRNA splicing factors, an important facet in the assembly of an mRNP. The fact that the arginine methylation of the Snp1 homolog U1-70K is conserved in higher eukaryotes suggests that the role of arginine methylation in regulating the spliceosome may also be conserved.