Protein arginine methylation of Npl3 promotes splicing of the SUS1 intron harboring non-consensus 5′ splice site and branch site

Highlights

• Yeast protein arginine methyltransferase Hmt1 regulates splicing via the SR-/hnRNP-like protein Npl3.

• Methylation of Npl3 facilitates the spliceosomal usage of consensus splice sites on yeast SUS1 intron 1.

• Hmt1 promotes optimal biochemical association between Npl3 and U1 snRNP via U1 snRNP subunit Mud1.

Abstract

Protein arginine methylation occurs on spliceosomal components and spliceosome-associated proteins, but how this modification contributes to their function in pre-mRNA splicing remains sparse. Here we provide evidence that protein arginine methylation of the yeast SR-/hnRNP-like protein Npl3 plays a role in facilitating efficient splicing of the SUS1 intron that harbors a non-consensus 5′ splice site and branch site. In yeast cells lacking the major protein arginine methyltransferase HMT1, we observed a change in the co-transcriptional recruitment of the U1 snRNP subunit Snp1 and Npl3 to pre-mRNAs harboring both consensus (ECM33 and ASC1) and non-consensus (SUS1) 5′ splice site and branch site. Using an Npl3 mutant that phenocopies wild-type Npl3 when expressed in Δhmt1 cells, we showed that the arginine methylation of Npl3 is responsible for this. Examination of pre-mRNA splicing efficiency in these mutants reveals the requirement of Npl3 methylation for the efficient splicing of SUS1 intron 1, but not of ECM33 or ASC1. Changing the 5′ splice site and branch site in SUS1 intron 1 to the consensus form restored splicing efficiency in an Hmt1-independent manner. Results from biochemical studies show that methylation of Npl3 promotes its optimal association with the U1 snRNP through its association with the U1 snRNP subunit Mud1. Based on these data, we propose a model in which Hmt1, via arginine methylation of Npl3, facilitates U1 snRNP engagement with the pre-mRNA to promote usage of non-consensus splice sites by the splicing machinery.

Introduction

Eukaryotic gene expression requires functional integration of splicing and transcription of precursor mRNAs (pre-mRNAs) [1], [2]. During pre-mRNA splicing, it is critical that introns are precisely removed from the precursor transcript via the spliceosome, a macromolecular RNA-protein complex whose recruitment and activities are controlled by cis sequence elements and by trans-acting factors [3], [4]. In Saccharomyces cerevisiae, five small nuclear ribonucleoproteins (snRNPs) comprise the core of the spliceosome. In the consensus model of spliceosome assembly, the U1 snRNP first recognizes the 5′ splice site in the pre-mRNA through base-pairing between the 5′-end of the U1 snRNA and the 5′ splice site sequence, forming the earliest commitment complex (CC1) [5], [6], [7]. The branch site is then recognized by the branchpoint binding protein (BBP/Msl5) and its binding partner Mud2 to form commitment complex 2 (CC2) [8], [9], [10]. The BBP-Mud2 heterodimer then interacts with the U1 snRNP, forming a bridge between the 5′ splice site and the branch site [11]. Prp5 and Sub2, both of which are DEAD/DECD box proteins, then promote displacement of BBP and Mud2 by the U2 snRNP at the branch site in an ATP-dependent manner, resulting in the pre-spliceosome or A complex [12], [13], [14]. The preformed U4/U6.U5 tri-snRNP then joins to form the assembled spliceosome [15], [16], [17], [18]. Following the binding of the tri-snRNP, major structural rearrangements occur within the spliceosome, resulting in the release of U1 and U4 snRNPs and formation of new base-pairings between U6 and the 5′ splice site and between U2 and U6 snRNA [19], [20]. A protein complex called the nineteen complex (NTC) then stabilizes the association of U5 and U6 with the spliceosome, thereby allowing the formation of an activated spliceosome to catalyze the actual splicing reaction [21]. The snRNPs and non-snRNP-associated factors are co-transcriptionally recruited through an ordered process [22], [23], [24]. Although splicing can occur post-transcriptionally, the coupling of this process to transcription likely maximizes its fidelity and efficiency [25].

While the basic components of the splicing machinery are conserved between S. cerevisiae and mammals, splicing regulators of the serine/arginine-rich (SR) protein family are present only in the latter [26]. Previous studies in mammalian cells showed that SR proteins stabilize interactions between the U1 snRNP and pre-mRNAs [27], [28]. It is thought that SR proteins bind to the exonic sequences adjacent to suboptimal splice sites, consequently promoting the recruitment of U1 and U2 snRNPs and thereby playing a significant role in constitutive splicing of pre-mRNAs as well as regulating alternative splicing. In yeast, most splice sites obey a strict consensus, and since only a handful of intron-containing genes possess more than a single intron it was thought that the lack of SR proteins in the budding yeast reflects lack of a need for alternative splicing [29]. Nevertheless, a number of yeast proteins such as Npl3, Hrb1, and Gbp2 possess characteristics of mammalian SR proteins [30]. Interestingly, Npl3 additionally shares characteristics with heterogeneous nuclear ribonucleoproteins (hnRNPs) – a large number of RGG boxes and RNA-recognition motifs (RRMs) [31], [32].

Several post-translational modifications have been implicated in the control of pre-mRNA splicing [reviewed in 33]. For example, ubiquitination of the DExD/H box family protein Brr2 suppresses its ability to unwind U4/U6 RNA duplex [34]. Recently, protein arginine methylation has been linked to the control of pre-mRNA splicing in several model systems: budding yeast [35], cultured mammalian cells [36], [37], plants [38], and mice [39]. Protein arginine methylation alters the biochemical properties of the side chain of the amino acid arginine [reviewed in 40]. This modification is catalyzed by members of the protein arginine methyltransferase (PRMT) family of enzymes, which are divided into four subtypes based on the specific type of methylarginine that is formed. The type I PRMTs transfer either one or two methyl groups from S-adenosyl-l-methionine (AdoMet) to a single guanidino nitrogen on a protein-incorporated arginine residue, thus forming monomethylarginine (MMA) or asymmetric dimethylarginine (aDMA), respectively. Type II PRMTs likewise catalyze monomethylation, but then add the second methyl group from AdoMet to the opposing guanidino nitrogen within the arginine residue, thereby forming symmetric dimethylarginine (sDMA). Sm proteins, which are critical to the assembly of functional U snRNPs in the cell, contain sDMAs that are modified by PRMT5, a type II PRMT [41], [42]. Arginine methylation has been shown to modulate alternative splicing in the higher eukaryotes. In humans, PRMT1-mediated methylation of RBM15 regulates alternative splicing of genes important for megakaryocyte differentiation [43]. PRMT4/CARM1 has been implicated in the regulation of alternative splicing and has been shown to methylate splicing factors U1C, CA150, SAP49 and SmB [44]. PRMT9 has also been shown to methylate splicing factor SAP145 and SF3B2, and is implicated in U2 snRNP maturation [45], [46]. PRMT2 interacts with splicing factors and regulates alternative splicing of the gene BCL-X [47]. Depletion of PRMT5 leads to reduced methylation of Sm proteins and aberrant alternative splicing of specific mRNAs in neural stem/progenitor cells [39]. In Arabidopsis thaliana, PRMT5 has been shown to regulate alternative splicing and this regulation is critical for the synchronization of the organism's circadian clock [38].

In S. cerevisiae, Hmt1 (also termed Rmt1) is the only known type I PRMT and it is the yeast homolog of mammalian PRMT1 [48], [49]. Hmt1 has been shown to methylate the SR-/hnRNP-like Npl3 [49], [50] and the vast majority of aDMAs are present within the RGG context [51]. Previously, we showed that Hmt1 methylates the U1 snRNP subunit Snp1 and that Hmt1 is required for proper co-transcriptional recruitment of specific snRNP subunits and snRNP-associated factors to their genomic targets [35]. In the current study, we demonstrate that Hmt1 plays a role in facilitating the spliceosome usage of non-consensus splice sites, and that it does so via methylation of the SR-/hnRNP-like protein Npl3. Our data show that Δhmt1 cells display altered recruitment of U1 snRNP subunit Snp1 and SR-like protein Npl3 to intron-containing genes SUS1, ECM33, and ASC1 in an RNA-dependent manner. However, only SUS1 intron 1, which harbors a non-consensus 5′ splice site and branch site, exhibits a decreased splicing efficiency in these cells. Using an Npl3 mutant that phenocopies wild-type Npl3 behavior when expressed in Δhmt1 cells, we show that the arginine methylation of Npl3 is responsible for the recruitment and splicing efficiency changes observed in Δhmt1 cells. Biochemical characterizations using methylated and unmethylated form of Npl3 indicate a methylation-dependent association between Npl3 and the U1 snRNP via Mud1, a subunit of U1 snRNP. Overall, our data support a model in which Hmt1, via methylating Npl3, promotes optimal U1 snRNP engagement with non-consensus 5′ splice site and branch site in SUS1 in order to achieve its efficient splicing.