The nuclear structures that contain symmetrical dimethylated arginine (sDMA)–modified proteins and the role of this posttranslational modification is unknown. Here we report that the Cajal body is a major epitope in HeLa cells for an sDMA-specific antibody and that coilin is an sDMA-containing protein as analyzed by using the sDMA-specific antibody and matrix-assisted laser desorption ionization time of flight mass spectrometry. The methylation inhibitor 5′-deoxy-5′-methylthioadenosine reduces the levels of coilin methylation and causes the appearance of SMN-positive gems. In cells devoid of Cajal bodies, such as primary fibroblasts, sDMA-containing proteins concentrated in speckles. Cells from a patient with spinal muscular atrophy, containing low levels of the methyl-binding protein SMN, localized sDMA-containing proteins in the nucleoplasm as a discrete granular pattern. Splicing reactions are efficiently inhibited by using the sDMA-specific antibody or by using hypomethylated nuclear extracts, showing that active spliceosomes contain sDMA polypeptides and suggesting that arginine methylation is important for efficient pre-mRNA splicing. Our findings support a model in which arginine methylation is important for the localization of coilin and SMN in Cajal bodies.

The mammalian protein arginine methyltransferase 3 (PRMT3) catalyzes the formation of asymmetric (type I) dimethylarginine in vitro. As yet, natural substrates and cellular pathways modulated by PRMT3 remain unknown. Here, we have identified an ortholog of PRMT3 in fission yeast. Tandem affinity purification of fission yeast PRMT3 coupled with mass spectrometric protein identification revealed that PRMT3 associates with components of the translational machinery. We identified the 40S ribosomal protein S2 as the first physiological substrate of PRMT3. In addition, a fraction of yeast and human PRMT3 cosedimented with free 40S ribosomal subunits, as determined by sucrose gradient velocity centrifugation. The activity of PRMT3 is not essential since prmt3-disrupted cells are viable. Interestingly, cells lacking PRMT3 showed an accumulation of free 60S ribosomal subunits resulting in an imbalance in the 40S:60S free subunits ratio; yet prerRNA processing appeared to occur normally. Our results identify PRMT3 as the first type I ribosomal protein arginine methyltransferase and suggest that it regulates ribosome biosynthesis at a stage beyond pre-rRNA processing.

Hmt1 is the major type I arginine methyltransferase in the yeastIn eukaryotic cells, pre-messenger RNAs (pre-mRNAs) must be fully processed and packaged into mature messenger ribonucleoparticles (mRNPs) before export to the cytoplasm as fully translatable mRNAs. Intranuclear RNA processing steps, such as 5Ј-capping, splicing, 3Ј-end cleavage, and polyadenylation, are accomplished through the association of numerous RNA-binding proteins (RBPs) such as serine/arginine-rich (SR) proteins and heterogeneous nuclear ribonucleoproteins (hnRNPs) with the pre-mRNA (for review, see Dreyfuss et al. 2002;Lei and Silver 2002b;Reed and Hurt 2002). Many RBPs that participate in RNA processing and export contain a variety of posttranslational modifications such as phosphorylation and methylation. The dynamic interactions between RBPs and pre-mRNAs suggest that their binding to and dissociation from RNAs and other proteins may be regulated by these posttranslational modifications.One type of posttranslational modification commonly found in RNA-binding proteins is the methylation of arginine residues, usually in the context of arginine-and glycine-rich motifs (for review, see Gary and Clarke 1998). The enzymes that catalyze this process are called protein arginine methyltransferase, or PRMTs. hnRNPs are a major substrate of PRMT1 in yeast and mammalian cells. Methylated hnRNPs contain at least one N-terminal RRM-type (RNA recognition motif) RNA-binding motif in conjunction with RGG-rich (arginine-glycineglycine) repeats, the sites of arginine methylation, in the C-terminal domains (Liu and Dreyfuss 1995).Recent studies have shown that arginine methylation is important for modulating protein-protein interactions. For example, loss of arginine methylation on the STAT1 protein inhibits association with its inhibitor PIAS, resulting in decreased interferon responses mediated by STAT1 (Mowen et al. 2001). Methylation of the Src kinase substrate Sam68 has been shown to change its affinity for SH3-containing proteins, resulting in alteration of its function (Bedford et al. 2000). In addition, arginine methylation of the transcriptional elongation factor Spt5 regulates its interaction with RNA polymerase (Pol) II, thereby globally affecting transcription (Kwak et al. 2003). However, the precise role of methylation of the many RBPs remains unclear.

The poly(A)-binding protein nuclear 1 (PABPN1) is a ubiquitously expressed protein that is thought to function during mRNA poly(A) tail synthesis in the nucleus. Despite the predicted role of PABPN1 in mRNA polyadenylation, little is known about the impact of PABPN1 deficiency on human gene expression. Specifically, it remains unclear whether PABPN1 is required for general mRNA expression or for the regulation of specific transcripts. Using RNA sequencing (RNA–seq), we show here that the large majority of protein-coding genes express normal levels of mRNA in PABPN1–deficient cells, arguing that PABPN1 may not be required for the bulk of mRNA expression. Unexpectedly, and contrary to the view that PABPN1 functions exclusively at protein-coding genes, we identified a class of PABPN1–sensitive long noncoding RNAs (lncRNAs), the majority of which accumulated in conditions of PABPN1 deficiency. Using the spliced transcript produced from a snoRNA host gene as a model lncRNA, we show that PABPN1 promotes lncRNA turnover via a polyadenylation-dependent mechanism. PABPN1–sensitive lncRNAs are targeted by the exosome and the RNA helicase MTR4/SKIV2L2; yet, the polyadenylation activity of TRF4-2, a putative human TRAMP subunit, appears to be dispensable for PABPN1–dependent regulation. In addition to identifying a novel function for PABPN1 in lncRNA turnover, our results provide new insights into the post-transcriptional regulation of human lncRNAs.

Poly(A)-binding proteins (PABPs) are important to eukaryotic gene expression. In the nucleus, the PABP PABPN1 is thought to function in polyadenylation of pre-mRNAs. Deletion of fission yeast pab2, the homolog of mammalian PABPN1, results in transcripts with markedly longer poly(A) tails, but the nature of the hyperadenylated transcripts and the mechanism that leads to RNA hyperadenylation remain unclear. Here we report that Pab2 functions in the synthesis of noncoding RNAs, contrary to the notion that PABPs function exclusively on protein-coding mRNAs. Accordingly, the absence of Pab2 leads to the accumulation of polyadenylated small nucleolar RNAs (snoRNAs). Our findings suggest that Pab2 promotes poly(A) tail trimming from pre-snoRNAs by recruiting the nuclear exosome. This work unveils a function for the nuclear PABP in snoRNA synthesis and provides insights into exosome recruitment to polyadenylated RNAs.

Telomere maintenance by the telomerase reverse transcriptase requires a noncoding RNA subunit that acts as a template for the synthesis of telomeric repeats. In humans, the telomerase RNA (hTR) is a non-polyadenylated transcript produced from an independent transcriptional unit. As yet, the mechanism and factors responsible for hTR 3' end processing have remained largely unknown. Here, we show that hTR is matured via a polyadenylation-dependent pathway that relies on the nuclear poly(A)-binding protein PABPN1 and the poly(A)-specific RNase PARN. Depletion of PABPN1 and PARN results in telomerase RNA deficiency and the accumulation of polyadenylated precursors. Accordingly, a deficiency in PABPN1 leads to impaired telomerase activity and telomere shortening. In contrast, we find that hTRAMP-dependent polyadenylation and exosome-mediated degradation function antagonistically to hTR maturation, thereby limiting telomerase RNA accumulation. Our findings unveil a critical requirement for RNA polyadenylation in telomerase RNA biogenesis, providing alternative approaches for telomerase inhibition in cancer.

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Telomerase is a specialized reverse transcriptase (RT) that is minimally composed of a protein catalytic subunit and an RNA component. The RNA subunit contains a short template sequence that directs the synthesis of DNA repeats at the ends of chromosomes. Human telomerase activity can be reconstituted in vitro by the expression of the human telomerase protein catalytic subunit (hTERT) in the presence of recombinant human telomerase RNA (hTR) in a rabbit reticulocyte lysate (RRL) system. We analyzed telomerase activity and binding of hTR to hTERT in RRL by expressing different hTERT and hTR variants. hTRs containing nucleotide substitutions that are predicted to disrupt base pairing in the P3 helix of the pseudoknot weakly reconstituted human telomerase activity yet retained their ability to bind hTERT. Our results also identified two distinct regions of hTR that can independently bind hTERT in vitro. Furthermore, sequences or structures between nucleotides 208 and 330 of hTR (which include the conserved CR4-CR5 domain) were found to be important for hTERT-hTR interactions and for telomerase activity reconstitution. Human TERT carboxyterminal amino acid deletions extending to motif E or the deletion of the first 280 amino acids abolished human telomerase activity without affecting the ability of hTERT to associate with hTR, suggesting that the RT and RNA binding functions of hTERT are separable. These results indicate that the reconstitution of human telomerase activity in vitro requires regions of hTERT that (i) are distinct from the conserved RT motifs and (ii) bind nucleotides distal to the hTR template sequence.