Aaron J. Shatkin

5'-Terminal structure and mRNA stability.

Reovirus mRNAs with 5′-terminal m7GpppGm or GpppG are more stable than mRNA containing unblocked ppG 5′-ends when injected into Xenopus laevis oocytes or incubated in cell-free protein synthesising extracts of wheat germ and mouse L cells. The greater stability of mRNA with blocked 5′ termini is not dependent upon translation but seems to result from protection against 5′-exonucleolytic degradation.

Viral and cellular mRNA capping: Past and prospects

Publisher Summary This chapter focuses on the history of the discovery of cap and an update of research on viral and cellular-messenger RNA (mRNA) capping. Cap structures of the type m7 GpppN(m)pN(m)p are present at the 5′ ends of nearly all eukaryotic cellular and viral mRNAs. A cap is added to cellular mRNA precursors and to the transcripts of viruses that replicate in the nucleus during the initial phases of transcription and before other processing events, including internal N6A methylation, 3′-poly (A) addition, and exon splicing. Despite the variations on the methylation theme, the important biological consequences of a cap structure appear to correlate with the N7-methyl on the 5′-terminal G and the two pyrophosphoryl bonds that connect m7G in a 5′–5′ configuration to the first nucleotide of mRNA. In addition to elucidating the biochemical mechanisms of capping and the downstream effects of this 5′- modification on gene expression, the advent of gene cloning has made available an ever-increasing amount of information on the proteins responsible for producing caps and the functional effects of other cap-related interactions. Genetic approaches have demonstrated the lethal consequences of cap failure in yeasts, and complementation studies have shown the evolutionary functional conservation of capping from unicellular to metazoan organisms.

The ends of the affair: Capping and polyadenylation

Nearly all mRNAs are post-transcriptionally modified at their 5′ and 3′ ends, by capping and polyadenylation, respectively. These essential modifications are of course chemically quite distinct, as are the enzymatic complexes responsible for their synthesis. But recent studies have uncovered some similarities as well. For example, both involve entirely protein machinery, which is now the exception rather than the rule in RNA processing and modification reactions, and the two reactions share one important factor, namely RNA polymerase II. In this brief review, we describe progress in understanding the enzymes and factors that participate in these two processes, highlighting the evolutionary conservation, from yeast to humans, that has become apparent.

High-resolution structures of HIV-1 reverse transcriptase/TMC278 complexes: Strategic flexibility explains potency against resistance mutations.

TMC278 is a diarylpyrimidine (DAPY) nonnucleoside reverse transcriptase inhibitor (NNRTI) that is highly effective in treating wild-type and drug-resistant HIV-1 infections in clinical trials at relatively low doses (∼25–75 mg/day). We have determined the structure of wild-type HIV-1 RT complexed with TMC278 at 1.8 Å resolution, using an RT crystal form engineered by systematic RT mutagenesis. This high-resolution structure reveals that the cyanovinyl group of TMC278 is positioned in a hydrophobic tunnel connecting the NNRTI-binding pocket to the nucleic acid-binding cleft. The crystal structures of TMC278 in complexes with the double mutant K103N/Y181C (2.1 Å) and L100I/K103N HIV-1 RTs (2.9 Å) demonstrated that TMC278 adapts to bind mutant RTs. In the K103N/Y181C RT/TMC278 structure, loss of the aromatic ring interaction caused by the Y181C mutation is counterbalanced by interactions between the cyanovinyl group of TMC278 and the aromatic side chain of Y183, which is facilitated by an ∼1.5 Å shift of the conserved Y183MDD motif. In the L100I/K103N RT/TMC278 structure, the binding mode of TMC278 is significantly altered so that the drug conforms to changes in the binding pocket primarily caused by the L100I mutation. The flexible binding pocket acts as a molecular “shrink wrap” that makes a shape complementary to the optimized TMC278 in wild-type and drug-resistant forms of HIV-1 RT. The crystal structures provide a better understanding of how the flexibility of an inhibitor can compensate for drug-resistance mutations.

Cap and cap-binding proteins in the control of gene expression

The 5′ mRNA cap structure is essential for efficient gene expression from yeast to human. It plays a critical role in all aspects of the life cycle of an mRNA molecule. Capping occurs co‐transcriptionally on the nascent pre‐mRNA as it emerges from the RNA exit channel of RNA polymerase II. The cap structure protects mRNAs from degradation by exonucleases and promotes transcription, polyadenylation, splicing, and nuclear export of mRNA and U‐rich, capped snRNAs. In addition, the cap structure is required for the optimal translation of the vast majority of cellular mRNAs, and it also plays a prominent role in the expression of eukaryotic, viral, and parasite mRNAs. Cap‐binding proteins specifically bind to the cap structure and mediate its functions in the cell. Two major cellular cap‐binding proteins have been described to date: eukaryotic translation initiation factor 4E (eIF4E) in the cytoplasm and nuclear cap binding complex (nCBC), a nuclear complex consisting of a cap‐binding subunit cap‐binding protein 20 (CBP 20) and an auxiliary protein cap‐binding protein 80 (CBP 80). nCBC plays an important role in various aspects of nuclear mRNA metabolism such as pre‐mRNA splicing and nuclear export, whereas eIF4E acts primarily as a facilitator of mRNA translation. In this review, we highlight recent findings on the role of the cap structure and cap‐binding proteins in the regulation of gene expression. We also describe emerging regulatory pathways that control mRNA capping and cap‐binding proteins in the cell. WIREs RNA 2011 2 277–298 DOI: 10.1002/wrna.52

Origin of splice junction phosphate in tRNAs processed by HeLa cell extract

Abstract Two cloned tRNA genes that contain intervening sequences, yeast tRNA UCG Ser and Xenopus laevis tRNA Tyr , were transcribed in HeLa cell extract. Precursor tRNAs were formed, and were converted to spliced products by a process of excision-ligation. The novel sequences resulting from ligation of tRNA half-molecules were examined by fingerprinting and nearest neighbor analyses. The results indicate that during tRNA splicing in HeLa cell extract, the 3′-terminal phosphate of the 5′ half-molecule is incorporated into a normal 3′,5′-phosphodiester linkage that forms the splice junction. This ligation pathway in HeLa cell extract is distinct from the one described previously in wheat germ extract, which involves formation of 2′-phosphomonoester, 3′,5′-phosphodiester linkage with the 3′,5′-bond derived from a 5′-terminal phosphate.

Sequence homology between the structural polypeptides of minute virus of mice.

The DNA-containing (full) particles of minute virus of mice contain three polypeptide species, designated A (Mr=83,000), B (Mr=64,000) and C (Mr=61,000). These three proteins are compared here by tryptic and chymotryptic fingerprinting after radio-iodination of their tyrosyl residues in vitro. Polypeptide B and C digests are almost identical, thus confirming the precursor-product relationship between them suggested by previous kinetic studies. Both types of fingerprint show one peptide which occurs in the C polypeptide but not in B, indicating that the cleavage in vivo may not occur at either a tryptic or chymotryptic site. In addition to the relationship between B and C, all of the sequence of B is present in the largest polypeptide A, which constitutes≈16% of the total virion protein. The A polypeptide contains additional tyrosyl peptides, comprising about 20% of the total, which do not occur in either B or C. Proteolytic digestion of intact full particles in vitro shows that the cleavage of B in vivo can be closely mimicked by trypsin and to a lesser extent by chymotrypsin. However, the B polypeptide in the empty virion is resistant to cleavage by either enzyme in vitro, indicating that it adopts a different conformation in each particle type. This correlates well with the in vivo observation that empty particles contain polypeptide B, in addition to A, and do not contain any polypeptide C. The A polypeptide is completely resistant to cleavage by either enzyme in either particle, suggesting that the conformation of the common sequence in polypeptide A may be similar to that adopted by polypeptide B in empty virions. A scheme for the maturation of infectious parvovirus virions is described.

Crystal engineering of HIV-1 reverse transcriptase for structure-based drug design

HIV-1 reverse transcriptase (RT) is a primary target for anti-AIDS drugs. Structures of HIV-1 RT, usually determined at ∼2.5–3.0 Å resolution, are important for understanding enzyme function and mechanisms of drug resistance in addition to being helpful in the design of RT inhibitors. Despite hundreds of attempts, it was not possible to obtain the structure of a complex of HIV-1 RT with TMC278, a nonnucleoside RT inhibitor (NNRTI) in advanced clinical trials. A systematic and iterative protein crystal engineering approach was developed to optimize RT for obtaining crystals in complexes with TMC278 and other NNRTIs that diffract X-rays to 1.8 Å resolution. Another form of engineered RT was optimized to produce a high-resolution apo-RT crystal form, reported here at 1.85 Å resolution, with a distinct RT conformation. Engineered RTs were mutagenized using a new, flexible and cost effective method called methylated overlap-extension ligation independent cloning. Our analysis suggests that reducing the solvent content, increasing lattice contacts, and stabilizing the internal low-energy conformations of RT are critical for the growth of crystals that diffract to high resolution. The new RTs enable rapid crystallization and yield high-resolution structures that are useful in designing/developing new anti-AIDS drugs.

Recombinant Human mRNA Cap Methyltransferase Binds Capping Enzyme/RNA Polymerase IIo Complexes

Guanine N-7 methylation is an essential step in the formation of the m7GpppN cap structure that is characteristic of eukaryotic mRNA 5′ ends. The terminal 7-methylguanosine is recognized by cap-binding proteins that facilitate key events in gene expression including mRNA processing, transport, and translation. Here we describe the cloning, primary structure, and properties of human RNA (guanine-7-)methyltransferase. Sequence alignment of the 476-amino acid human protein with the corresponding yeast ABD1 enzyme demonstrated the presence of several conserved motifs known to be required for methyltransferase activity. We also identified a Drosophila open reading frame that encodes a putative RNA (guanine-7-)methyltransferase and contains these motifs. Recombinant human methyltransferase transferred a methyl group fromS-adenosylmethionine to GpppG 5′ends, which are formed on RNA polymerase II transcripts by the sequential action of RNA 5′-triphosphatase and guanylyltransferase activities in the bifunctional mammalian capping enzyme. Binding studies demonstrated that the human cap methyltransferase associated with recombinant capping enzyme. Consistent with selective capping of RNA polymerase II transcripts, methyltransferase also formed ternary complexes with capping enzyme and the elongating form of RNA polymerase II.

The Tat/TAR-dependent phosphorylation of RNA polymerase II C-terminal domain stimulates cotranscriptional capping of HIV-1 mRNA

The HIV type 1 (HIV-1) Tat protein stimulates transcription elongation by recruiting P-TEFb (CDK9/cyclin T1) to the transactivation response (TAR) RNA structure. Tat-induced CDK9 kinase has been shown to phosphorylate Ser-5 of RNA polymerase II (RNAP II) C-terminal domain (CTD). Results presented here demonstrate that Tat-induced Ser-5 phosphorylation of CTD by P-TEFb stimulates the guanylyltransferase activity of human capping enzyme and RNA cap formation. Sequential phosphorylation of CTD by Tat-induced P-TEFb enhances the stimulation of human capping enzyme guanylyltransferase activity and RNA cap formation by transcription factor IIH-mediated CTD phosphorylation. Using an immobilized template assay that permits isolation of transcription complexes, we show that Tat/TAR-dependent phosphorylation of RNAP II CTD stimulates cotranscriptional capping of HIV-1 mRNA. Upon transcriptional induction of latently infected cells, accumulation of capped transcripts occurs along with Ser-5-phosphorylated RNAP II in the promoter proximal region of the HIV-1 genome. Therefore, these observations suggest that Tat/TAR-dependent phosphorylation of RNAP II CTD is crucial not only in promoting transcription elongation but also in stimulating nascent viral RNA capping.