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A general method for large scale preparation of uniformly isotopically labeled ribonucleotides and RNAs is described. Bacteria are grown on isotopic growth medium, and their nucleic acids are harvested and degraded to mononucleotides. These are enzymatically converted into ribonucleoside triphosphates, which are used in transcription reactions in vitro to prepare RNAs for NMR studies. For 15N-labeling, E.coli is grown on 15N-ammonium sulfate, whereas for 13C-labeling, Methylophilus methylotrophus is grown on 13C-methanol, which is more economical than 13C-glucose. To demonstrate the feasibility and utility of this method, uniformly 13C-labeled ribonucleotides were used to synthesize a 31 nucleotide HIV TAR RNA that was analyzed by 3D-NMR. This method should find widespread use in the structural analysis of RNA by NMR.

La is a conserved eukaryotic RNA-binding protein best known for its role in the biogenesis of noncoding RNAs transcribed by RNA polymerase III. To broaden our understanding of the function of the La homologous protein (Lhp1) in Saccharomyces cerevisiae, we have taken a genomics approach. Lhp1 ribonucleoprotein complexes were immunoprecipitated and bound RNAs were examined by hybridization to whole-genome microarrays that include >6,000 ORFs, documented noncoding RNAs, and the intervening intergenic regions. Demonstrating the validity of this approach, associations with previously known Lhp1p-associated RNAs were detected and associations with additional noncoding RNAs, including multiple tRNAs and small nucleolar RNAs, were revealed. Indicating that this approach provides a robust method for discovering RNAs, the data also identify associations between Lhp1p and several intergenic regions, three of which encode the recently annotated putative snoRNAs: RUF1, RUF2, and RUF3. Unexpectedly, we find that Lhp1p is also associated with a subset of coding mRNAs. These mRNAs include many ribosomal protein transcripts as well as the mRNA encoding Hac1p, a transcription factor required during the unfolded protein stress response. In cells lacking LHP1, Hac1p levels are decreased 2-to 3-fold, whereas no changes are detected in the levels of spliced or unspliced HAC1 mRNA or in the stability of Hac1p. Finally, although LHP1 is dispensable for growth under standard conditions, we find that it is required when the unfolded protein response is induced at elevated temperatures. These results suggest that Lhp1p may play a novel role in the translation of one or more cellular mRNAs. L a is an abundant eukaryotic RNA-binding protein implicated in multiple steps of RNA metabolism, including transcription and 3Ј end processing of RNA polymerase III (RNA pol III) transcripts, as well as translation of certain viral and endogenous mRNAs containing internal ribosome entry site (IRES) sequences (1). The best characterized role of La is in the biogenesis and processing of a variety of noncoding RNAs (1). La binds the 3Ј terminal UUU OH sequences of newly synthesized RNA pol III transcripts such as pre-tRNAs and pre-5S rRNA and protects them from degradation (2, 3). In yeast, La homologous protein (LHP1) is nonessential, which is surprising, given that its metazoan counterparts are involved in processing such a wide range of essential, noncoding RNAs. Nevertheless, genetic and biochemical analyses in Saccharomyces cerevisiae have confirmed that Lhp1p is involved in the processing of newly synthesized RNA pol III transcripts (4, 5), and revealed a similar role with noncoding RNAs generated by RNA pol II, spliceosomal small nuclear RNAs (snRNAs) (6), and U3 small nucleolar RNA (snoRNA) (7). Lhp1p is known to bind these noncoding RNA precursors and is thought to facilitate their maturation by stabilizing them from digestion. Because Lhp1p is not required for maintaining normal levels of mature ribonucleoproteins, it has been suggested that Lh...

J proteins are structurally diverse, obligatory cochaperones of Hsp70s, each with a highly conserved J domain that plays a critical role in the stimulation of Hsp70's ATPase activity. The essential protein, Cwc23, is one of 13 J proteins found in the cytosol and/or nucleus of Saccharomyces cerevisiae. We report that a partial loss-of-function CWC23 mutant has severe, global defects in pre-mRNA splicing. This mutation leads to accumulation of the excised, lariat form of the intron, as well as unspliced pre-mRNA, suggesting a role for Cwc23 in spliceosome disassembly. Such a role is further supported by the observation that this mutation results in reduced interaction between Cwc23 and Ntr1 (SPP382), a known component of the disassembly pathway. However, Cwc23 is a very atypical J protein. Its J domain, although functional, is dispensable for both cell viability and pre-mRNA splicing. Nevertheless, strong genetic interactions were uncovered between point mutations encoding alterations in Cwc23's J domain and either Ntr1 or Prp43, a DExD/H-box helicase essential for spliceosome disassembly. These genetic interactions suggest that Hsp70-based chaperone machinery does play a role in the disassembly process. Cwc23 provides a unique example of a J protein; its partnership with Hsp70 plays an auxiliary, rather than a central, role in its essential cellular function.Hsp70-based machineries constitute a key component of the cell's chaperone network, playing a central role in many processes, including de novo protein folding, protein translocation across membranes, and remodeling of protein complexes (6, 13). By their ability to bind to short, exposed, hydrophobic stretches of polypeptide, Hsp70s serve as the core of this protein folding machinery. However, Hsp70s cannot function alone. J proteins (often referred to as Hsp40s) are their obligatory partners, serving to stimulate Hsp70's ATPase activity and thereby stabilizing interaction with client proteins (19). J proteins are very diverse but, by definition, contain an ϳ70-amino-acid "J domain" that interacts directly with the Hsp70 ATPase domain (12,17). It is well established that the J domain is critical for function of the Hsp70-based chaperone machinery, since single amino acid alterations in its highly conserved HPD motif disrupt function, both in vitro and in vivo, without affecting domain structure (9, 14). The Saccharomyces cerevisiae genome encodes 22 J proteins. Of these, 13 are found in the cytosol and/or nucleus. One, Cwc23, is the focus of this report. Deletion of CWC23 causes inviability in some strain backgrounds, although adaptation or suppression allows slow growth in others (31, 34). The growth defect of cwc23⌬ cells cannot be rescued by overexpression of any of the other 12 J proteins (31), indicating that Cwc23 carries out a specialized cellular function. Cwc23 has been linked to premRNA splicing via both genome-wide and spliceosomal component-directed physical interaction studies (7,25).Pre-mRNA splicing is a highly precise and stepwise process wher...

Writing is an essential part of a successful career in science. As such, many undergraduate science courses have begun to implement writing assignments that reflect "real-world" applications and focus on a critical analysis of current literature; these assignments are often in the form of a review or a research proposal. The semester-long project described herein is a unique marriage of these two ideas: students first select a topic and conduct a literature review, and then choose an area of that same topic to investigate further in a peer-reviewed grant proposal. A modified version of this project, which incorporates peer-reviewed oral presentations, is also discussed. This project is designed for an upper-level undergraduate course, typically having 15−20 students, and the approach (or parts of the approach) has been successfully incorporated in an advanced organic chemistry course, a biochemistry capstone course, and courses in endocrinology, as well as ecophysiology.