Yasuhiro Furuichi

Effect of 5′-terminal structure and base composition on polyribonucleotide binding to ribosomes

Abstract Ribopolymers of variable base composition and 5′-terminal structure were synthesized with polynucleotide phosphorylase. Under primer-dependent conditions, m 7 GpppGmpC (m 7 G-cap) † , its alkali-treated m 7 G ring-opened derivative, GpppGpC and ppGpC but not m 7 GpppGmpCp, m 7 GpppGm or GpppG were incorporated as 5′-termini. The ribopolymers were compared with reovirus mRNA, which contains m 7 G-cap, for their ability to form initiation complexes with wheat germ 40 S ribosomal subunits and 80 S ribosomes. The presence of 5′-terminal m 7 G was required for stable complex formation by some ribopolymers while for others binding was increased by two- to fourfold. The final level of binding observed was similar to that with reovirus mRNA. In addition to 5′-terminal m 7 G, the base composition of the ribopolymers markedly influenced binding. Some ribopolymers including m 7 G-cap (A) n did not bind significantly; m 7 G-cap (U) n formed 40 S complexes while m 7 G-cap (A,U) n bound to 80 S ribosomes. The ribopolymer m 7 G-cap (A 2 ,U 2 ,G) n directed protein synthesis as measured by amino acid incorporation into polypeptides, methionine tRNA association with 40 S complexes, and puromycin reactivity of 80 S-associated methionine and, like reovirus mRNA, its binding to ribosomes was inhibited by 7-methylguanosine 5′-monophosphate.

Reovirus transcriptase and capping enzymes are active in intact virions.

Abstract Expression of reovirus-associated enzymes was detected in purified virions. As reported previously for chymotrypsin-derived viral cores, untreated virions catalyzed the synthesis of short oligonucleotides corresponding to the 5′-terminal sequence of viral mRNAS. Oligonucleotide synthesis by both kinds of particles required a divalent cation, and Mn+2 was more effective than Mg+2. The transcriptase activity in virions, in contrast to viral cores, was apparently incapable of elongating nascent chains. Consequently, virions produced no viral mRNAs during incubation in vitro for several hours. Transcriptase activity as measured by oligonucleotide synthesis was highly temperature dependent in virions and cores (∼51° optimum) and similar to mRNA synthesis by cores. The several other reovirus-associated enzymes involved in 5′-terminal cap formation were also active in virions. They include nucleotide phosphohydrolase, guanylyltransferase and methyltransferase activities. The results indicate that elongation, but not initiation-related transcription events, is prevented in reovirions by structural constraints on the transcriptase/template RNA complex.

5′-termini of reovirus mRNA: Ability of viral cores to form caps post-transcriptionally

Abstract The 5′-terminal “cap” structure of reovirus mRNA, m 7 GpppG m -C, is synthesized by five coordinate reactions that involve the sequential action of the virion-associated RNA polymerase, nucleotide phosphohydrolase, guanylyltransferase, and two distinct methyltransferases. Caps are synthesized in vitro by viral cores at an early stage of transcription. However, under appropriate conditions, cap formation can also occur post-transcriptionally. Preformed, nascent mRNA with 5′-terminal ppG-C was modified to m 7 GpppG m -C by guanylyl- and methyltransferases under conditions that prevented further RNA synthesis and chain completion, indicating that the transferases and RNA polymerase activities are not tightly coupled within cores. Similarly, the polymerase appears to be independent of the RNA 5′-modifying enzymes, since transcription occurred in the presence of the imido analog of GTP, GMP-PNHP, yielding RNA with a 5′-terminal triphosphate structure, p(NH)ppG-C. The imido linkage of the β- and γ-phosphates was not cleaved by the nucleotide phosphohydrolase, consequently preventing the formation of diphosphate-containing 5′-termini required for the synthesis of GpppG-C by guanylate transfer. In contrast to its inactivity as a GMP acceptor, GMP-PNHP was utilized as a GMP donor for conversion of ppG-C termini to GpppG-C by reovirus cores, consistent with the proposed mechanism of cap synthesis.

Gene mapping of cytoplasmic polyhedrosis virus of silkworm by the full-length mRNA prepared under optimized conditions of transcription in vitro.

Viral mRNA synthesis by the RNA polymerase associated with purified cytoplasmic polyhedrosis virus (CPV) was studied. The formation of full-length mRNA products was facilitated by including in the reaction mixture 100 mM sodium acetate, high concentrations of ribonucleoside triphosphates, and proteinase K. The 10 different species of CPV mRNAS were resolved into 9 discrete RNA bands by agarose gel electrophoresis at pH 3.5 in buffer containing 7 M urea. Each purified viral mRNA hybridized specifically to one of the viral genome segments which were separated by polyacrylamide gel electrophoresis into the 10 species of dsRNA. The relationship between the genome segments and their cognate mRNAs synthesized in vitro is thus established. Under optimal conditions of mRNA synthesis each of the genome segments was transcribed at a similar rate as determined from the yield of individual separated mRNA species. A recycling model of genome-associated RNA polymerase for viral transcription is discussed.

5′-TERMINAL 7-METHYLGUANOSINE IN VIRAL mRNAs AND ITS ROLE IN TRANSLATION

ABSTRACT. Many eukaryotic cellular and viral mRNAs contain blocked, methylated 5′-terminal “caps” of the general type m 7 G(5′)ppp(5′)N. The prevalence of 5′-terminal m 7 G in eukaryotic mRNA is consistent with its role in translation. Methylated reo and VSV mRNAs are 20-fold more actively translated in protein synthesizing extracts of wheat germ as compared to the unmethylated mRNAs. The m 7 G in caps is the important constituent for messenger activity and its removal from reovirus and globin mRNA by β-elimination results in a concomitant decrease in ability to direct polypeptide synthesis. The requirement for m 7 G occurs at or before the binding of mRNA to 40S ribosomal subunits. The methylated cap comprises part of the binding sequence of reovirus mRNA since 40S ribosomal subunits confer 80–100% protection of caps against RNase digestion. Upon conversion to 80S initiation complexes, the cap remains protected with only some messenger species. The size of the protected 5′-terminal fragments also varies for different reovirus mRNA species. Those fragments that retain the cap after RNase treatment of mRNA-ribosome complexes rebind to ribosomes to a greater extent than uncapped fragments. In addition to m 7 G, ribopolymer binding to ribosomes is influenced by base composition and possibly sequence. Certain synthetic ribopolymers (e.g. poly U, A·U, A·U·G) bind to a limited extent to 40S subunits when they contain 5′-terminal GpppG, ppG, pN, or ring-opened m 7 G Δ pppG; addition of the cap increases their binding. Others (e.g. poly C, poly A·G, poly C·G) do not bind even when capped. Capped poly A·U or poly A·U·G, but not poly U, can also form 80S complexes under conditions of reo mRNA translation. Mechanisms of m 7 G-dependent recognition of mRNA during initiation of protein synthesis will be discussed.

SEPARATION OF FULL LENGTH TRANSCRIPTS AND GENOME RNA PLUS AND MINUS STRANDS FROM CYTOPLASMIC POLYHEDROSIS VIRUS OF BOMBYX MORI

ABSTRACT Under optimal conditions, CPV transcription in vitro was maintained for more than 24 hr, resulting in large quantities of full-size mRNAs. The mRNAs were resolved into nine discrete bands by agarose gel electrophoresis in the presence of 7 M urea. The genome dsRNA segments coding for each mRNA were identified by hybridizing the separated, 32P-labeled mRNA to a mixture of genome RNAs. Each of the genome segments appears to be transcribed at the same rate, producing equal quantities by weight but different molar amounts of the separated individual mRNA species. These results imply that each of the genome segments is transcribed repeatedly by an equal number of template-associated RNA polymerases at the same rate of chain elongation. The genome RNAs of CPV and reovirus, labeled at the 3′-termini with 32pCp by RNA ligase, were separated by agarose gel electrophoresis into the plus and minus strands. The RNA strands of plus polarity were found to migrate faster in the gel than those of minus polarity for all CPV dsRNA genome segments, whereas the opposite was seen for most of the reovirus genome segments. The basis of this separation is due to retention of secondary structure during the electrophoresis even in the presence of 7 M urea.

5′-CAPPING AND EUKARYOTIC mRNA FUNCTION

Publisher Summary This chapter focuses on 5′-capping and eukaryotic mRNA function. Studies of mRNA structure and synthesis have revealed unanticipated complexities in the organization and expression of eukaryotic genes. In contrast to their prokaryotic counterparts, most eukaryotic mRNAs share several distinctive structural features. In cellular mRNAs and mRNAs of viruses that replicate in the nucleus of infected cells, caps are present on nuclear messenger precursors but not on ribosomal or tRNA transcripts. In some mRNAs, caps may be synthesized as part of the initiation of transcription and involved in its regulation. The chapter highlights that capped mRNAs are degraded more slowly than the corresponding uncapped mRNAs in HeLa cell nuclear and cytoplasmic fractions, in cell-free protein synthesizing systems from wheat germ and mouse L cells, and in microinjected Xenopus oocytes. The chapter also discusses in detail about aspects of cap structure, distribution among eukaryotic mRNAs, mechanisms of synthesis and functional effects on mRNA stability, and initiation of translation. Recent experimental results concerning the influence of cap on the initiation of eukaryotic transcription and translation have also been presented.