Eukaryotic cells contain several unconventional poly(A) polymerases in addition to the canonical enzymes responsible for the synthesis of poly(A) tails of nuclear messenger RNA precursors. The yeast protein Trf4p has been implicated in a quality control pathway that leads to the polyadenylation and subsequent exosome-mediated degradation of hypomethylated initiator tRNAMet (tRNAi Met). Here we show that Trf4p is the catalytic subunit of a new poly(A) polymerase complex that contains Air1p or Air2p as potential RNA-binding subunits, as well as the putative RNA helicase Mtr4p. Comparison of native tRNAi Met with its in vitro transcribed unmodified counterpart revealed that the unmodified RNA was preferentially polyadenylated by affinity-purified Trf4 complex from yeast, as well as by complexes reconstituted from recombinant components. These results and additional experiments with other tRNA substrates suggested that the Trf4 complex can discriminate between native tRNAs and molecules that are incorrectly folded. Moreover, the polyadenylation activity of the Trf4 complex stimulated the degradation of unmodified tRNAi Met by nuclear exosome fractions in vitro. Degradation was most efficient when coupled to the polyadenylation activity of the Trf4 complex, indicating that the poly(A) tails serve as signals for the recruitment of the exosome. This polyadenylation-mediated RNA surveillance resembles the role of polyadenylation in bacterial RNA turnover.

Eukaryotic mRNA precursors (pre-mRNAs) 1 are synthesized and processed in the nucleus prior to their export to the cytoplasm where they serve as templates for protein synthesis. Transcription is coupled spatially and temporally to the capping of the pre-mRNA at the 5Ј-end, splicing, and 3Ј-end formation. The mature 3Ј-ends of most eukaryotic mRNAs are generated by endonucleolytic cleavage of the primary transcript followed by the addition of a poly(A) tail to the upstream cleavage product (for reviews see Refs. 1 and 2). In mammals, these reactions are catalyzed by a large multicomponent complex that is assembled in a cooperative manner on specific cis-acting sequence elements in the pre-mRNA. The cleavage and polyadenylation specificity factor (CPSF) (3) recognizes the highly conserved hexanucleotide AAUAAA, whereas the cleavage stimulation factor (CstF) (4) binds a more degenerate GUor U-rich element downstream of the poly(A) site. It has been suggested that in vivo CPSF and CstF may become associated with each other prior to pre-mRNA binding, recognizing the two elements in a concerted manner (5). In addition, the cleavage reaction requires mammalian cleavage factor I (CF I m ), cleavage factor II m (CF II m ), and poly(A) polymerase (PAP). After the first step of 3Ј-end processing, CPSF remains bound to the upstream cleavage fragment and tethers PAP to the 3Ј-end of the pre-mRNA (6). In the presence of the nuclear poly(A)-binding protein 1 (PABPN1), PAP elongates the poly(A) tail in a processive manner (6). These factors are both necessary and sufficient to reconstitute cleavage and polyadenylation in vitro. However, the other proteins involved in either transcription, such as the C-terminal domain of RNA polymerase II, or capping (nuclear cap-binding complex) and splicing (U2AF65) have been shown to greatly enhance the efficiency of the first step of the reaction (7-9).Three major polypeptides of 25, 59, and 68 kDa and one minor polypeptide of 72 kDa copurify with CF I m activity from HeLa cell nuclear extract (10). Reconstitution of CF I m activity with recombinant proteins suggests that CF I m is a heterodimer consisting of the 25-kDa subunit and one of the larger polypeptides (11). All of the three larger proteins appear to be highly related in their amino acid sequence. Moreover, all of the CF I m subunits are only present in metazoan organisms. The primary sequence of the 25-kDa polypeptide contains a NUDIX motif (12). The amino acid composition of the 68-kDa protein has a domain organization that is reminiscent of spliceosomal SR proteins. Members of the SR family of splicing factors contain one or more N-terminal RNA recognition motifs (RRMs) that function in sequence-specific RNA binding and a C-terminal domain rich in alternating arginine and serine residues, referred to as RS domain, which is required for proteinprotein interactions with other RS domains (13). In the 68-kDa protein, the RRM and the RS-like domain are separated by a

WW domains are protein modules that mediate protein-protein interactions through recognition of prolinerich peptide motifs and phosphorylated serine/threonine-proline sites. To pursue the functional properties of WW domains, we employed mass spectrometry to identify 148 proteins that associate with 10 human WW domains. Many of these proteins represent novel WW domain-binding partners and are components of multiprotein complexes involved in molecular processes, such as transcription, RNA processing, and cytoskeletal regulation. We validated one complex in detail, showing that WW domains of the AIP4 E3 proteinubiquitin ligase bind directly to a PPXY motif in the p68 subunit of pre-mRNA cleavage and polyadenylation factor Im in a manner that promotes p68 ubiquitylation. The tested WW domains fall into three broad groups on the basis of hierarchical clustering with respect to their associated proteins; each such cluster of bound proteins displayed a distinct set of WW domain-binding motifs. We also found that separate WW domains from the same protein or closely related proteins can have different specificities for protein ligands and also demonstrated that a single polypeptide can bind multiple classes of WW domains through separate prolinerich motifs. These data suggest that WW domains provide a versatile platform to link individual proteins into physiologically important networks.Many signaling proteins contain modular domains that mediate specific protein-protein interactions, frequently through the recognition of short peptide motifs in their binding partners (56). In many cases these interactions are regulated by posttranslational modifications, such as phosphorylation. Interaction domains can thereby control the subcellular localization, enzymatic activity, and substrate specificity of regulatory proteins and the assembly of multiprotein complexes, and thus the flow of information through signaling pathways.WW domains comprise a family of protein-protein interaction modules that are found in many eukaryotes and are present in approximately 50 human proteins (6; see Fig. 1). Within these polypeptides, WW domains are joined to a number of distinct interaction modules, including phosphotyrosinebinding domains (i.e., in the FE65 protein) and FF domains (CA150 and FBP11), as well as protein localization domains, such as C2 (NEDD4 family proteins) and pleckstrin homology domains (PLEKHA5). WW domains are also linked to a variety of catalytic domains, including HECT E3 protein-ubiquitin ligase domains (in NEDD4 family proteins), rotomerase/peptidyl prolyisomerase domains (Pin1), and Rho GTPase-activating protein domains. Consequently, WW domain-containing proteins are involved in a variety of cellular processes, including transcription, RNA processing, protein trafficking, receptor signaling, and control of the cytoskeleton (32,33,68). WW domain-mediated interactions have been implicated in cancer (4, 75), in hereditary disorders, such as Liddle's syndrome (66) and Rett's syndrome (8), as well as in Alzheimer's (46, ...

The protein factor U2 snRNP Auxiliary Factor (U2AF) 65 is an essential component required for splicing and involved in the coupling of splicing and 3 0 end processing of vertebrate pre-mRNAs. Here we have addressed the mechanisms by which U2AF 65 stimulates pre-mRNA 3 0 end processing. We identify an arginine/serine-rich region of U2AF 65 that mediates an interaction with an RS-like alternating charge domain of the 59 kDa subunit of the human cleavage factor I (CF I m ), an essential 3 0 processing factor that functions at an early step in the recognition of the 3 0 end processing signal. Tethered functional analysis shows that the U2AF 65/CF I m 59 interaction stimulates in vitro 3 0 end cleavage and polyadenylation. These results therefore uncover a direct role of the U2AF 65/CF I m 59 interaction in the functional coordination of splicing and 3 0 end processing.

Mammalian cleavage factor I (CF I m ) is composed of two polypeptides of 25 kDa and either a 59 or 68 kDa subunit (CF I m 25, CF I m 59, CF I m 68). It is part of the cleavage and polyadenylation complex responsible for processing the 39 ends of messenger RNA precursors. To investigate post-translational modifications in factors of the 39 processing complex, we systematically searched for enzymes that modify arginines by the addition of methyl groups. Protein arginine methyltransferases (PRMTs) are such enzymes that transfer methyl groups from S-adenosyl methionine to arginine residues within polypeptide chains resulting in mono-or dimethylated arginines. We found that CF I m 68 and the nuclear poly(A) binding protein 1 (PABPN1) were methylated by HeLa cell extracts in vitro. By fractionation of these extracts followed by mass spectral analysis, we could demonstrate that the catalytic subunit PRMT5, together with its cofactor WD45, could symmetrically dimethylate CF I m 68, whereas pICln, the third polypeptide of the complex, was stimulatory. As sites of methylation in CF I m 68 we could exclusively identify arginines in a GGRGRGRF or ''GAR'' motif that is conserved in vertebrates. Further in vitro assays revealed a second methyltransferase, PRMT1, which modifies CF I m 68 by asymmetric dimethylation of the GAR motif and also weakly methylates the C-termini of both CF I m 59 and CF I m 68. The results suggest that native-as compared with recombinant-protein substrates may contain additional determinants for methylation by specific PRMTs. A possible involvement of CF I m methylation in the context of RNA export is discussed.

NS1, the major nonstructural protein of the parvovirus minute virus of mice, is a multifunctional phosphoprotein which is involved in cytotoxicity, transcriptional regulation, and initiation of viral DNA replication. For coordination of these various functions during virus propagation, NS1 has been proposed to be regulated by posttranslational modifications, in particular phosphorylation. Recent in vitro studies (J. P. F. Nüesch, R. Corbau, P. Tattersall, and J. Rommelaere, J. Virol. 72:8002–8012, 1998) provided evidence that distinct NS1 activities, notably the intrinsic helicase function, are modulated by the phosphorylation state of the protein. In order to study the dependence of the initiation of viral DNA replication on NS1 phosphorylation and to identify the protein kinases involved, we established an in vitro replication system that is devoid of endogenous protein kinases and is based on plasmid substrates containing the minimal left-end origins of replication. Cellular components necessary to drive NS1-dependent rolling-circle replication (RCR) were freed from endogenous serine/threonine protein kinases by affinity chromatography, and the eukaryotic DNA polymerases were replaced by the bacteriophage T4 DNA polymerase. While native NS1 (NS1P) supported RCR under these conditions, dephosphorylated NS1 (NS1O) was impaired. Using fractionated HeLa cell extracts, we identified two essential protein components which are able to phosphorylate NS1O, are enriched in protein kinase C (PKC), and, when present together, reactivate NS1O for replication. One of these components, containing atypical PKC, was sufficient to restore NS1O helicase activity. The requirement of NS1O reactivation for characteristic PKC cofactors such as Ca2+/phosphatidylserine or phorbol esters strongly suggests the involvement of this protein kinase family in regulation of NS1 replicative functions in vitro.

The parvovirus minute virus of mice NS1 protein is a multifunctional protein involved in a variety of processes during virus propagation, ranging from viral DNA replication to promoter regulation and cytotoxic action to the host cell. Since NS1 becomes phosphorylated during infection, it was proposed that the different tasks of this protein might be regulated in a coordinated manner by phosphorylation. Indeed, comparing biochemical functions of native NS1 with its dephosphorylated counterpart showed that site-specific nicking of the origin and the helicase and ATPase activities are remarkably reduced upon NS1 dephosphorylation while site-specific affinity of the protein to the origin became enhanced. As a consequence, the dephosphorylated polypeptide is deficient for initiation of DNA replication. By adding fractionated cell extracts to a kinase-free in vitro replication system, the combination of two protein components containing members of the protein kinase C (PKC) family was found to rescue the replication activity of the dephosphorylated NS1 protein upon addition of PKC cofactors. One of these components, termed HA-1, also stimulated NS1 helicase function in response to acidic lipids but not phorbol esters, indicating the involvement of atypical PKC isoforms in the modulation of this NS1 function (J. P. F. Nüesch, S. Dettwiler, R. Corbau, and J. Rommelaere, J. Virol. 72:9966–9977, 1998). The present study led to the identification of atypical PKCλ/ι as the active component of HA-1 responsible for the regulation of NS1 DNA unwinding and replicative functions. Moreover, a target PKCλ phosphorylation site was localized at S473 of NS1. By site-directed mutagenesis, we showed that this residue is essential for NS1 helicase activity but not promoter regulation, suggesting a possible modulation of NS1 functions by PKCλ phosphorylation at residue S473.