Arginine methylation and binding of Hrp1p to the efficiency element for mRNA 3'-end formation

Hrp1p is a heterogeneous ribonucleoprotein (hnRNP) from the yeast Saccharomyces cerevisiae that is involved in the cleavage and polyadenylation of the 3'-end of mRNAs and mRNA export. In addition, Hrplp is one of several RNA-binding proteins that are posttranslationally modified by methylation at arginine residues. By using functional recombinant Hrp1p, we have identified RNA sequences with specific high affinity binding sites. These sites correspond to the efficiency element for mRNA 3'-end formation, UAUAUA. To examine the effect of methylation on specific RNA binding, purified recombinant arginine methyltransferase (Hmt1p) was used to methylate Hrp1p. Methylated Hrp1p binds with the same affinity to UAUAUA-containing RNAs as unmethylated Hrpl p indicating that methylation does not affect specific RNA binding. However, RNA itself inhibits the methylation of Hrp1p and this inhibition is enhanced by RNAs that specifically bind Hrpl p. Taken together, these data support a model in which protein methylation occurs prior to protein-RNA binding in the nucleus.     

Control of cleavage site selection during mRNA 3'' end formation by a yeast hnRNP.

Endonucleolytic cleavage of pre-mRNAs is the first step during eukaryotic mRNA 3' end formation. It has been proposed that cleavage factors CF IA, CF IB and CF II are required for pre-mRNA 3' end cleavage in yeast. CF IB is composed of a single polypeptide, Nab4p/Hrp1p, which is related to the A/B group of metazoan heterogeneous nuclear ribonucleoproteins (hnRNPs) that function as antagonistic regulators of 5' splice site selection. Here, we provide evidence that Nab4p/Hrp1p is not required for pre-mRNA 3' end endonucleolytic cleavage. We show that CF IA and CF II devoid of Nab4p/Hrp1p are sufficient to cleave a variety of RNA substrates but that cleavage occurs at multiple sites. Addition of Nab4p/Hrp1p prevents these alternative cleavages in a concentration-dependent manner, suggesting an essential and conserved role for some hnRNPs in pre-mRNA cleavage site selection.     

A putative ubiquitin ligase required for efficient mRNA export differentially affects hnRNP transport

BACKGROUND: In the nucleus, mRNAs are bound by hnRNP proteins. A subset of hnRNP proteins shuttle between the nucleus and cytoplasm and are believed to promote mRNA export by acting as adaptors between mRNA and the transport machinery. The existence of multiple shuttling hnRNP proteins raises the question of whether differentially regulated, hnRNP-specific mRNA export pathways exist. RESULTS: We have determined that Tom1p, a conserved protein with a hect (homology to E6-AP carboxyl terminus) E3 ubiquitin ligase domain, is required for efficient mRNA export in S. cerevisiae, yet differentially affects hnRNP protein localization and export. Mutations in tom1 predicted to abolish ubiquitin ligase activity block efficient export of Nab2p and mRNA, causing Nab2p-mRNA complexes to accumulate in a punctate pattern coincident with the nuclear pore complex (NPC). Notably, the subcellular distribution of several other hnRNP proteins is not affected. In particular, Np13p remains mRNA-associated and continues to be efficiently exported in tom1 mutants. CONCLUSION: Our results demonstrate that mutations predicted to affect the enzymatic activity of the Tom1p ubiquitin ligase differentially affect export of hnRNP proteins in association with mRNA. We propose the existence of multiple mRNA export pathways, with export of Nab2p-associated mRNAs dependent on a branch of the ubiquitin protein modification pathway.     

Nuclear export of hnRNP Hrp1p and nuclear export of hnRNP Npl3p are linked and influenced by the methylation state of Npl3p

Eukaryotic mRNA processing and export are mediated by a series of complexes composed of heterogeneous nuclear ribonucleoproteins (hnRNPs). Many of these hnRNPs are methylated at arginine residues within their RGG domains. Although cellular arginine methylation is required for the efficient nuclear export of several hnRNPs, its role in this process is unknown. To address this question, we replaced the methylated RGG tripeptides of two hnRNPs, Npl3p and Hrp1p, with KGG. We found that these substitutions specifically abolish their methylation but have different effects on their nuclear export activity. Although the efficient export of Hrp1p requires cellular methyltransferase activity, the modification of Hrp1p itself is dispensable. In contrast, we found that Npl3 arginine methylation not only facilitates its own export but also is required for Hrp1p to efficiently exit the nucleus. Consistent with this observation, we found that Npl3p and Hrp1p exist in a ribonucleoprotein complex. We provide the first evidence that the arginine methylation of a particular protein directly affects its activity. Efficient export does not require methylation per se, but unmethylated arginine residues lead to retention of hnRNPs. Thus, arginine methylation serves to mask the Npl3p RGG domain for efficient ribonucleoprotein export.     

The Glc7p nuclear phosphatase promotes mRNA export by facilitating association of Mex67p with mRNA

mRNA export is mediated by Mex67p:Mtr2p/NXF1:p15, a conserved heterodimeric export receptor that is thought to bind mRNAs through the RNA binding adaptor protein Yra1p/REF. Recently, mammalian SR (serine/arginine-rich) proteins were shown to act as alternative adaptors for NXF1-dependent mRNA export. Npl3p is an SR-like protein required for mRNA export in S. cerevisiae. Like mammalian SR proteins, Npl3p is serine-phosphorylated by a cytoplasmic kinase. Here we report that this phosphorylation of Npl3p is required for efficient mRNA export. We further show that the mRNA-associated fraction of Npl3p is unphosphorylated, implying a subsequent nuclear dephosphorylation event. We present evidence that the essential, nuclear phosphatase Glc7p promotes dephosphorylation of Npl3p in vivo and that nuclear dephosphorylation of Npl3p is required for mRNA export. Specifically, recruitment of Mex67p to mRNA is Glc7p dependent. We propose a model whereby a cycle of cytoplasmic phosphorylation and nuclear dephosphorylation of shuttling SR adaptor proteins regulates Mex67p:Mtr2p/NXF1:p15-dependent mRNA export.