In vivo labelling of intermediates in the discontinuous synthesis of mRNAs in Trypanosoma brucei.

Discontinuous mRNA synthesis in trypanosomes is thought to involve a 140-nucleotide precursor, called the mini-exon-derived RNA or medRNA, which contributes its 5' 35 nucleotides to the 5' end of nascent mRNAs. We used in vivo labelling of RNA to show that medRNA has a half-life of less than 6 min, whereas putative high mol. wt intermediates containing the 3' part of the medRNA have an average half-life of less than 1 min. This eliminates priming of pre-mRNA synthesis by intact medRNA as the main mode of discontinuous mRNA synthesis. Potential intermediates of 35 and 105 nucleotides were labelled in parallel with medRNA, but their significance could not be assessed in RNA preparations containing medRNA, as they are also produced by artefactual cleavage of medRNA. We show, however, that high mol. wt RNA, free of medRNA, can release medRNA segments upon a debranching treatment. These results are consistent with a trans splicing mechanism involving short-lived forked intermediates, analogous to lariats in cis splicing systems.     

The cap of both miniexon-derived RNA and mRNA of trypanosomes is 7-methylguanosine.

Most, if not all, trypanosome mRNAs have the same 35-base sequence at their 5' terminus which is derived from a short RNA (medRNA) probably by the process of trans-splicing. It is of interest, evolutionarily and mechanistically, to determine the chemical structure of the 5' terminus of the precursor (medRNA) and product (mRNA). We demonstrate here that the cap structure of both is most probably 7-methylguanosine in a 5',5' triphosphate linkage, consistent with a precursor/product relationship.     

Direct analysis of the mini-exon donor RNA of Trypanosoma brucei: detection of a novel cap structure also present in messenger RNA.

The mini-exon, a short segment found at the 5' end of trypanosome mRNAs, is contributed by a small RNA, the mini-exon donor (medRNA). In vivo 32P-labeled medRNA, a set of smaller RNAs related to it, and mRNA, were purified from Trypanosoma brucei by hybrid selection and gel electrophoresis. Using RNA fingerprinting and sequencing techniques, mini-exon oligonucleotides were identified and characterized. We detected a novel 5' terminal capped oligonucleotide present in both medRNA and mRNA. This structure contained m7G and at least four modified nucleotides, not identified previously. If the T. brucei mini-exon has exactly four transcribed nucleotides upstream from its originally designated 5' end, it would begin with the sequence: m7GpppA*A*C*U*AA*CG (asterisks denote modification) and medRNA would be 140 nucleotides long, excluding the m7G residue. The mini-exon contains, and retains during its transfer to mRNA, a novel 5' terminal structure whose presence could confer unique functional attributes.     

Physical identification of branched intron side-products of splicing in Trypanosoma brucei.

Every mRNA in trypanosomes consists of two exons, a common 5' capped mini-exon or spliced leader and a coding-exon. All evidence suggests that the exons are joined by trans-splicing of two individual precursor RNAs, the mini-exon donor RNA or spliced leader precursor RNA (medRNA) and the pre-mRNA. We studied intermediates of the splicing reaction using denaturing two-dimensional PAGE and structurally identified a group of small (approximately 180-300 nt) non-polyadenylated, Y-shaped branched RNAs. The branched Y-shaped RNAs contain the 105 nt medRNA derived intron, joined in a 2'-5' phosphodiester bond to small heterogeneously sized RNAs. These non-polyadenylated branched Y-shaped RNA molecules are analogous to the lariat shaped introns of higher eukaryotes and presumably represent the released intron-like by-products of a trans-splicing reaction which joins the mini-exon and the major coding-exon.     

Regulated transport of messenger ribonucleic acid from isolated liver nuclei by nucleic acid binding proteins

Rat liver nucleocytosolic messenger ribonucleic acid (mRNA) transport is shown to be regulated by proteins with a high affinity for nucleic acids. In the cell-free system described, the energy-dependent transport of all RNA classes [transfer RNA (tRNA), mRNA, and ribosomal RNA (rRNA)] exhibited a dependence upon the availability of discrete minor sets of cytosol proteins. In addition to having a different level of saturation, only the mRNA "transport protein" activities are increased by adenosine cyclic 3',5'-phosphate (cAMP), an effect most likely mediated by a cAMP-dependent protein kinase. The mRNA transport proteins were isolated from cytosol by precipitation with streptomycin sulfate followed by deoxyribonucleic acid (DNA)-cellulose affinity chromatography, or from oligo-(thymidylate)-cellulose bound cytoplasmic messenger ribonucleoprotein (mRNP) particles by high-salt extraction. Either method yielded a protein fraction which exhibited a 1000-fold increase in mRNA transport activity as compared to cytosol. Over one-half of the mRNA transport activity is associated with the mRNP of the cell. A partial homology between the cytosol and mRNP-derived proteins was demonstrated by polyacrylamide gel electrophoresis. One major (20 000 daltons) and several minor proteins (23 000, 52 000, 54 000, and 72 000 daltons) were in common. Nuclear 4-5S exited from in vitro incubated nuclei in three phases, according to their differential in vivo rates of labeling and intranuclear pool sizes. The amount of nuclear RNA transported in vitro as mRNA (about 1.0%) agrees wtih the in vivo estimates. Additional evidence for in vivo equivalence was provided by the physicochemical characterization and bioassay of the RNA. The transported mRNA sedimented in urea-sucrose gradients as an 8-18S heterodisperse product. This RNA initiated cell-free translation with the synthesis of precursor peptides as diverse in size as those for albumin and alpha 2U-globulin. The relative abundancies of various transported mRNAs were different than the corresponding abundancies of liver cytoplasmic mRNAs.     

Purification and properties of a new bacillus subtilis RNA processing enzyme. Cleavage of phage SP82 mRNA and Bacillus subtilis precursor rRNA

An RNA processing activity capable of cleaving Bacillus subtilis phage SP82 early mRNA has been purified to apparent homogeneity from crude extracts of uninfected B. subtilis. The enzyme, a functional monomer of Mr approximately 27,000, cleaves only at the 5' side of adenosine residues at processing sites and is competitively inhibited by double-stranded synthetic RNA polymers. Processed SP82 mRNAs were translated in an Escherichia coli cell-free system and no qualitative or quantitative effects of processing on the synthesis of polypeptides was observed. The processing enzyme does not cleave T7 mRNA, E. coli precursor rRNA, or double-stranded poly(AU). A recombinant plasmid containing portions of two B. subtilis rRNA gene sets was transcribed in vitro and the resulting RNA was cleaved in the spacer region between the 16 S and 23 S rRNA genes. The ability of the B. subtilis processing enzyme to cleave SP82 mRNA and B. subtilis precursor rRNA and the fact that the enzyme has high affinity for double-stranded RNA suggest that it is the functional analog of E. coli RNase III.     

Identification of a protein complex that binds to a dodecamer sequence found at the 3'' ends of yeast mitochondrial mRNAs.

An activity from Saccharomyces cerevisiae mitochondria was identified that specifically bound to a 12-nucleotide sequence, AAUAA(U/C)AUUCUU, that is a site for processing of pre-mRNAs so as to generate the mature 3' ends of mRNAs. Because processing occurs 3' to the end of the dodecamer site, all mRNAs in yeast mitochondria terminate with that sequence. RNase T1 digestion fragments which terminated precisely at their 3' ends with the dodecamer sequence bound the activity, indicating that mRNAs in vivo would be capable of binding. Gel mobility shift analyses using RNA oligonucleotides showed that binding was reduced by a U-to-A substitution at position 3 of the dodecamer sequence; a C-to-A substitution at position 10 eliminated binding. UV cross-linking identified three polypeptides with approximate molecular masses of 19, 60, and 70 kDa as constituents of the binding activity. These estimates included the contribution of the 32P-labeled RNA oligonucleotide used to tag these polypeptides. An oligonucleotide with a UA-->AU substitution at positions 3 and 4 of the dodecamer site formed complexes deficient in the 19-kDa species, suggesting that binding specificity was inherent to the higher-molecular-weight polypeptides. Assembly of the complex at a dodecamer site on an RNA protected sequences located 5' to the dodecamer site from digestion by a nucleoside triphosphate-dependent 3' exoribonuclease found in yeast mitochondria. Since mitochondrial mRNAs terminate with an intact dodecamer sequence, the binding activity may function in the stabilization of mRNAs in addition to 3'-end formation of mRNAs.     

Sesbania mosaic virus (SeMV) infectious clone: possible mechanism of 3' and 5' end repair and role of polyprotein processing in viral replication

Sesbania mosaic virus (SeMV) is a positive stranded RNA virus belonging to the genus Sobemovirus. Construction of an infectious clone is an essential step for deciphering the virus gene functions in vivo. Using Agrobacterium based transient expression system we show that SeMV icDNA is infectious on Sesbania grandiflora and Cyamopsis tetragonoloba plants. The efficiency of icDNA infection was found to be significantly high on Cyamopsis plants when compared to that on Sesbania grandiflora. The coat protein could be detected within 6 days post infiltration in the infiltrated leaves. Different species of viral RNA (double stranded and single stranded genomic and subgenomic RNA) could be detected upon northern analysis, suggesting that complete replication had taken place. Based on the analysis of the sequences at the genomic termini of progeny RNA from SeMV icDNA infiltrated leaves and those of its 3' and 5' terminal deletion mutants, we propose a possible mechanism for 3' and 5' end repair in vivo. Mutation of the cleavage sites in the polyproteins encoded by ORF 2 resulted in complete loss of infection by the icDNA, suggesting the importance of correct polyprotein processing at all the four cleavage sites for viral replication. Complementation analysis suggested that ORF 2 gene products can act in trans. However, the trans acting ability of ORF 2 gene products was abolished upon deletion of the N-terminal hydrophobic domain of polyprotein 2a and 2ab, suggesting that these products necessarily function at the replication site, where they are anchored to membranes.