The exon junction complex component Y14 modulates the activity of the methylosome in biogenesis of spliceosomal small nuclear ribonucleoproteins

Abstract

The RNA-binding protein Y14 heterodimerizes with Mago as the core of the exon junction complex during precursor mRNA splicing and plays a role in mRNA surveillance in the cytoplasm. Using the Y14/Magoh heterodimer as bait in a screening for its interacting partners, we identified the protein-arginine methyltransferase PRMT5 as a candidate. We show that Y14 and Magoh, but not other factors of the exon junction complex, interact with the cytoplasmic PRMT5-containing methylosome. We further provide evidence that Y14 promoted the activity of PRMT5 in methylation of Sm proteins of the small nuclear ribonucleoprotein core, whereas knockdown of Y14 reduced their methylation level. Moreover, Y14 overexpression induced the formation of a large, active, and small nuclear ribonucleoprotein (snRNP)-associated methylosome complex. However, Y14 may only transiently associate with the snRNP assembly complex in the cytoplasm. Together, our results suggest that Y14 facilitates Sm protein methylation probably by its activity in promoting the formation or stability of the methylosome-containing complex. We hypothesize that Y14 provides a regulatory link between pre-mRNA splicing and snRNP biogenesis.

FIGURE 1.

Y14 interacts with PRMT5 and the cytoplasmic methylosome. A, HeLa cell cytoplasmic extract was subjected to a pulldown assay using GST or GST-Y14/His-Magoh heterodimer (YM) as bait. Pulled down proteins were detected by silver staining of SDS-polyacrylamide gels (upper panel) and immunoblotting with anti-PRMT5 (lower panel). Lane M, molecular mass markers. B, HeLa cell cytoplasmic (Cyto.) or nuclear (Nucl.) extract was subjected to GST pulldown assays as in A. Immunoblotting was performed using antibodies against PRMT5, pICln, and MEP50. C, the pulldown assay was performed as in A. The bait proteins were as indicated above the gel. For both B and C, the bait proteins were detected by Coomassie (Cooma.) Brilliant Blue staining (lower panels). A schematic diagram shows the domain structure of Y14 and its truncated versions. RRM, RNA recognition motif; a.a., amino acids.

FIGURE 2.

Y14, but not other EJC factors, interacts with the methylosome. In all panels, HEK293 cells were transiently transfected with the empty vector (−) or the expression vector for each FLAG-tagged protein as indicated above the gels. Cell lysates (input) and anti-FLAG immunoprecipitates (IP) were analyzed by immunoblotting using antibodies against PRMT5, pICln, and FLAG.

FIGURE 3.

Y14 is not methylated by PRMT5 but may modulate its methylation activity in vitro. A, GST-Y14/His-Magoh (YM, non-phosphorylated; pYM, phosphorylated) and GST-SmD1 were subjected to the in vitro methylation assay using recombinant GST-PRMT1 or immunopurified PRMT5 in the presence of ³H-labeled methyl donor and then detected by autoradiography (Auto-radio.) and Coomassie (Cooma.) Brilliant Blue staining. The lower panel shows Coomassie Blue staining of purified GST-PRMT1 (lane 1) and immunopurified FLAG-PRMT5 (lane 2). Lane 3 shows immunoblotting (IB) of the FLAG-PRMT5 co-precipitate using antibodies against PRMT5, pICln, and MEP50. The asterisk in lane 1 indicates a truncated Y14 fragment. B, in vitro methylation of GST-SmD1 using FLAG-PRMT5 immunoprecipitates was performed as in A; different amounts of GST-Y14/His-Magoh were used. Upper panel, autoradiogram; lower panel, Coomassie Blue staining.

FIGURE 4.

Y14 modulates methylation activity of PRMT5 in vivo. Total cell lysates were prepared from the following cells: mock or FLAG-Y14-expressing HEK293 cells (A), HEK293 cells that were mock-transfected or transfected with luciferase (Luc), Y14-targeting siRNAs, or the FLAG-Y14 expression vector (B), and mock or FLAG-Y14-expressing HEK293 cells that were either additionally transfected or not with an siRNA targeting PRMT5 (si-PRMT5) (C). Antibodies used for immunoblotting were as indicated to the left of each gel. The SYM10 and Y12 antibodies recognize symmetrical dimethyl-arginine.

FIGURE 5.

Y14 promotes formation of larger PRMT5-containing complex. A, cytoplasmic extracts prepared from mock, FLAG-Y14-, or DDX3-expressing stable HEK293 cells were fractionated on a 5–20% sucrose gradient. Odd-numbered fractions were analyzed by immunoblotting with antibodies against PRMT5, pICln, transportin (TRN), or FLAG epitope. B, pooled 25 S fractions from mock cell extract (lanes 1 and 3) or FLAG-Y14-expressing extract (lanes 2 and 4) were subjected to immunoprecipitation (IP) by using Y12 antibody. Total proteins (lanes 1 and 2) and Y12 co-precipitates (lanes 3 and 4) were immunoblotted using anti-PRMT5 and anti-SNRPB. C, as in B, the 25 S fractions were subjected to cross-linking in the absence (−) or presence (+) of glutaraldehyde (GA) followed by immunoblotting using anti-PRMT5. D, in vitro methylation of GST-SmD1 was performed in the 25 S fractions using ³H-labeled methyl donor. GST-SmD1 was recovered and shown by autoradiography (Auto-radio.) (upper panel) and Coomassie (Cooma.) Blue staining (lower panel). Mo, monomer; Mu, multimer; Di, dimer.

FIGURE 6.

Y14 associates with snRNP assembly complex. HEK293 cells were mock-transfected or transiently transfected with the expression vector FLAG-Y14 alone (B, D, and F) or together with the vector of HA-SmB (A). Anti-FLAG immunoprecipitates (IP) were subjected to immunodetection of tagged proteins (A) or endogenous proteins as indicted (D and F) or Northern blotting using antisense RNA probes complementary to U1, U2, U4, U5, and U6 (B). In B, subcellular fractionation was evaluated by immunoblotting using anti-PARP1 and anti-α-tubulin (lower panel). N and C represent nuclear and cytoplasmic fractions, respectively. C, the FLAG-REF and -Y14 expression vectors were transiently transfected into HEK293 cells. Proteins that co-precipitated with anti-trimethylguanosine were immunoblotted with anti-FLAG. In D, anti-Gemin3 cross-reacted to a protein band indicated by the asterisk. E, HEK293 cells were mock-transfected (lane 1) or were transfected with vector expressing FLAG-PRMT5 (lane 2), HA-Y14 alone (lane 4), or both protein-expressing vectors (lane 3). Immunoprecipitation was performed using anti-FLAG followed by immunoblotting using antibodies as indicated.

FIGURE 7.

Model for function of Y14/Magoh complex in snRNP core methylation and assembly. The Y14/Magoh complex directly interacts with the methylosome to facilitate its activity in methylation of Sm proteins of the spliceosomal snRNPs and also promotes association of the SMN complex with the methylosome for snRNP core assembly. Subsequently, Y14/Magoh dissociates from the snRNPs before their import into the nucleus.