The tRNA m 1 A58 methyltransferase is composed of two subunits encoded by the essential genes TRM6 and TRM61 (formerly GCD10 and GCD14). The trm6-504 mutation results in a defective m 1 A methyltransferase (Mtase) and a temperature-sensitive growth phenotype that is attributable to the absence of m 1 A58 and consequential tRNA i Met instability. We used a genetic approach to identify the genes responsible for tRNA i Met degradation in trm6 cells. Three recessive extragenic mutations that suppress trm6-504 mutant phenotypes and restore hypomodified tRNA i Met to near normal levels were identified. The wild-type allele of one suppressor, DIS3/RRP44, encodes a 3-5 exoribonuclease and a member of the multisubunit exosome complex. We provide evidence that a functional nuclear exosome is required for the degradation of tRNA i The relatively unstable nature of messenger RNAs fueled the discovery of pathways that control the degradation of normal and abnormal mRNAs in the nucleus and cytoplasm (Hilleren and Parker 1999;Mitchell and Tollervey 2001;Wilusz et al. 2001;Maquat 2002;Moore 2002;Long and McNally 2003). Two general pathways of mRNA decay have been characterized in the yeast Saccharomyces cerevisiae, and homologs of most of the yeast proteins involved in mRNA turnover have been identified in metazoans. The first pathway initially requires shortening of the mRNA polyadenylate tail, followed by removal of the 5Ј cap structure (Wilusz et al. 2001), which leaves the body of the mRNA susceptible to 5Ј-3Ј exonucleolytic degradation by Xrn1p. The second pathway involves deadenylation of mRNAs and the 3Ј-5Ј degradation of the body of the mRNA by the exosome (Jacobs et al. 1998;Burkard and Butler 2000;van Hoof et al. 2000b;van Hoof and Parker 2002;Mitchell and Tollervey 2003).The exosome is a multisubunit complex of proteins with multiple functions in the processing, degradation, and retention of stable and unstable RNAs in the nucleus and cytoplasm. The cytoplasmic exosome directly interacts with Ski7p (Araki et al. 2001) and recruits the Ski2p, Ski3p, and Ski8p complex to the 3Ј end of a deadenylated mRNA (Brown et al. 2000) or an mRNA that is stalled on the ribosome because it lacks a stop codon (Jacobs et al. 1998;van Hoof et al. 2000b), and in turn each is degraded in a 3Ј-to-5Ј direction. In the nucleus, the exosome has been implicated in elimination of by-products of rRNA processing (ETS sequence). The nuclear exosome possesses an exonuclease, Rrp6p, not found in the cytoplasmic form (Allmang et al. 1999b). A specialized function of Rrp6p and the nuclear exosome appears to be in retaining mRNAs incorrectly processed at their 3Ј ends at the site of transcription to prevent their release into the cytoplasm (Hilleren et al. 2001;Libri et al. 2002). Thus far, the exosome has not been implicated in the destruction of stable RNAs that are rendered unstable due to mutations or defects in processing.

Protein kinase GCN2 regulates translation in amino acid-starved cells by phosphorylating elF2. GCN2 contains a regulatory domain related to histidyl-tRNA synthetase (HisRS) postulated to bind multiple deacylated tRNAs as a general sensor of starvation. In accordance with this model, GCN2 bound several deacylated tRNAs with similar affinities, and aminoacylation of tRNAphe weakened its interaction with GCN2. Unexpectedly, the C-terminal ribosome binding segment of GCN2 (C-term) was required in addition to the HisRS domain for strong tRNA binding. A combined HisRS+ C-term segment bound to the isolated protein kinase (PK) domain in vitro, and tRNA impeded this interaction. An activating mutation (GCN2c-E803V) that weakens PK-C-term association greatly enhanced tRNA binding by GCN2. These results provide strong evidence that tRNA stimulates the GCN2 kinase moiety by preventing an inhibitory interaction with the bipartite tRNA binding domain.

Gcd10p andMet maturation. The chromatographic behavior of elongator and initiator tRNA Met on a RPC-5 column indicated that both species are altered structurally in gcd10⌬ cells, and analysis of base modifications revealed that 1-methyladenosine (m 1 A) is undetectable in gcd10⌬ tRNA. Interestingly, gcd10 and gcd14 mutations had no effect on processing or accumulation of elongator tRNA Met , which also contains m 1 A at position 58, suggesting a unique requirement for this base modification in initiator maturation.

Recent studies of mRNA export factors have provided additional evidence for a mechanistic link between mRNA 3¢-end formation and nuclear export. Here, we identify Nab2p as a nuclear poly(A)-binding protein required for both poly(A) tail length control and nuclear export of mRNA. Loss of NAB2 expression leads to hyperadenylation and nuclear accumulation of poly(A) + RNA but, in contrast to mRNA export mutants, these defects can be uncoupled in a nab2 mutant strain. Previous studies have implicated the cytoplasmic poly(A) tail-binding protein Pab1p in poly(A) tail length control during polyadenylation. Although cells are viable in the absence of NAB2 expression when PAB1 is overexpressed, Pab1p fails to resolve the nab2D hyperadenylation defect even when Pab1p is tagged with a nuclear localization sequence and targeted to the nucleus. These results indicate that Nab2p is essential for poly(A) tail length control in vivo, and we demonstrate that Nab2p activates polyadenylation, while inhibiting hyperadenylation, in the absence of Pab1p in vitro. We propose that Nab2p provides an important link between the termination of mRNA polyadenylation and nuclear export.

Structural and mechanistic studies show that when the selection criteria of the immune system are changed, catalytic antibodies that have the efficiency of natural enzymes evolve, but the catalytic antibodies are much more accepting of a wide range of substrates. The catalytic antibodies were prepared by reactive immunization, a process whereby the selection criteria of the immune system are changed from simple binding to chemical reactivity. This process yielded aldolase catalytic antibodies that approximated the rate acceleration of the natural enzyme used in glycolysis. Unlike the natural enzyme, however, the antibody aldolases catalyzed a variety of aldol reactions and decarboxylations. The crystal structure of one of these antibodies identified the reactive lysine residue that was selected in the immunization process. This lysine is deeply buried in a hydrophobic pocket at the base of the binding site, thereby accounting for its perturbed pKa.

The combination of Reverse Transcription (RT) and high-throughput sequencing has emerged as a powerful combination to detect modified nucleotides in RNA via analysis of either abortive RT-products or of the incorporation of mismatched dNTPs into cDNA. Here we simultaneously analyze both parameters in detail with respect to the occurrence of N-1-methyladenosine (m1A) in the template RNA. This naturally occurring modification is associated with structural effects, but it is also known as a mediator of antibiotic resistance in ribosomal RNA. In structural probing experiments with dimethylsulfate, m1A is routinely detected by RT-arrest. A specifically developed RNA-Seq protocol was tailored to the simultaneous analysis of RT-arrest and misincorporation patterns. By application to a variety of native and synthetic RNA preparations, we found a characteristic signature of m1A, which, in addition to an arrest rate, features misincorporation as a significant component. Detailed analysis suggests that the signature depends on RNA structure and on the nature of the nucleotide 3′ of m1A in the template RNA, meaning it is sequence dependent. The RT-signature of m1A was used for inspection and confirmation of suspected modification sites and resulted in the identification of hitherto unknown m1A residues in trypanosomal tRNA.

A variety of nuclear ribonucleoproteins are believed to associate directly with nascent RNA polymerase II transcripts and remain associated during subsequent nuclear RNA processing reactions, including pre-mRNA polyadenylation and splicing as well as nucleocytoplasmic mRNA transport. To investigate the functions of these proteins by using a combined biochemical and genetic approach, we have isolated nuclear polyadenylated RNA-binding (NAB) proteins from Saccharomyces cerevisiae. Living yeast cells were irradiated with UV light to covalently cross-link proteins intimately associated with RNA in vivo. Polyadenylated RNAs were then selectively purified, and the covalent RNA-protein complexes were used to elicit antibodies in mice. Both monoclonal and polyclonal antibodies which detect a variety of NAB proteins were prepared. Here we characterize one of these proteins, NAB2. NAB2 is one of the major proteins associated with nuclear polyadenylated RNA in vivo, as detected by UV light-induced cross-linking. Cellular immunofluorescence, using both monoclonal and polyclonal antibodies, demonstrates that the NAB2 protein is localized within the nucleus. The deduced primary structure of NAB2 indicates that it is composed of at least two distinct types of RNA-binding motifs: (i) an RGG box recently described in a variety of heterogeneous nuclear RNA-, pre-rRNA-, mRNA-, and small nucleolar RNA-binding proteins and (ii) CCCH motif repeats related to the zinc-binding motifs of the largest subunit of RNA polymerases I, II, and III. In vitro RNA homopolymer/ single-stranded DNA binding studies indicate that although both the RGG box and CCCH motifs bind poly(G), poly(U), and single-stranded DNA, the CCCH motifs also bind to poly(A). NAB2 is located on chromosome VII within a cluster of ribonucleoprotein genes, and its expression is essential for cell growth.Gene expression is regulated at multiple levels following the initiation of transcription by RNA polymerase II. Nascent pre-mRNA transcripts are extensively modified to generate mRNAs (59). These modifications, all of which occur in the nucleus, may include 5'-end capping (46, 52), 3'-end cleavage and polyadenylation (64), splicing (27, 53), and modification of individual bases (16). All of these activities occur while pre-mRNAs are associated with a set of nuclear factors which may include both heterogeneous nuclear ribonucleoproteins (hnRNPs) and small nuclear ribonucleoproteins (snRNPs) (21,23,30,61). Once formed in the nucleus, mRNAs are transported into the cytoplasm by an unknown mechanism (40). mRNA turnover in both the nucleus and cytoplasm may also dramatically influence the amount of each type of mRNA available for translation (18).Although considerable information exists concerning the functional role of snRNPs in nuclear RNA processing (27, 30, 53, 61), very little is known about the specific functions of individual hnRNPs. Previous work focused on the idea that hnRNPs are primarily pre-mRNA-packaging proteins functionally analogous to the role that histones play...

Only five of the nine subunits of human eukaryotic translation initiation factor 3 (eIF3) have recognizable homologs encoded in the Saccharomyces cerevisiae genome, and only two of these (Prt1p and Tif34p) were identified previously as subunits of yeast eIF3. We purified a polyhistidine-tagged form of Prt1p (His-Prt1p) by Ni 2؉ affinity and gel filtration chromatography and obtained a complex of Ϸ600 kDa composed of six polypeptides whose copurification was completely dependent on the polyhistidine tag on His-Prt1p. All five polypeptides associated with His-Prt1p were identified by mass spectrometry, and four were found to be the other putative homologs of human eIF3 subunits encoded in S. cerevisiae: YBR079c/Tif32p, Nip1p, Tif34p, and YDR429c/Tif35p. The fifth Prt1p-associated protein was eIF5, an initiation factor not previously known to interact with eIF3. The purified complex could rescue Met-tRNA i Met binding to 40S ribosomes in defective extracts from a prt1 mutant or extracts from which Nip1p had been depleted, indicating that it possesses a known biochemical activity of eIF3. These findings suggest that Tif32p, Nip1p, Prt1p, Tif34p, and Tif35p comprise an eIF3 core complex, conserved between yeast and mammals, that stably interacts with eIF5. Nip1p bound to eIF5 in yeast two-hybrid and in vitro protein binding assays. Interestingly, Sui1p also interacts with Nip1p, and both eIF5 and Sui1p have been implicated in accurate recognition of the AUG start codon. Thus, eIF5 and Sui1p may be recruited to the 40S ribosomes through physical interactions with the Nip1p subunit of eIF3.The initiation of protein synthesis in eukaryotic cells is dependent on multiple initiation factors (eIFs) that stimulate the binding of mRNA and methionyl-initiator tRNA (tRNA i Met ) to 40S ribosomes to form the 48S preinitiation complex (39). The Met-tRNA i Met is delivered to 40S ribosomes in a ternary complex with eIF2 and GTP, whereas the binding of mRNA to ribosomes is stimulated by eIF4F, eIF4A, eIF4B (39), and the poly(A)-binding protein Pab1p (54). Joining of the 60S subunit to form an 80S initiation complex requires hydrolysis of the GTP bound to eIF2, dissociation of the ternary complex, and release of the eIF2-GDP binary complex, and eIF5 promotes these events by stimulating GTP hydrolysis on ternary complexes bound to 40S ribosomes (39).Mammalian eIF3 is a multisubunit complex that has been implicated in several aspects of 48S complex formation. The purified factor promotes dissociation of 80S ribosomes into 40S and 60S subunits, forming a complex with the 40S subunits, and stabilizes binding of the eIF2-GTP-Met-tRNA i Met ternary complex to the 40S ribosome. It also stimulates binding of mRNA to 40S subunits (9, 56), presumably through its interactions with the cap-binding initiation factor eIF4F (36, 38) or eIF4B (41). A mammalian eIF3 complex, purified by its ability to promote methionylpuromycin (Met-puromycin) synthesis by an 80S initiation complex in an assay containing purified eIF1A, eIF2, eIF5, eIF5A, and ribos...