The common 5'' terminal sequence on trypanosome mRNAs: a target for anti-messenger oligodeoxynucleotides.

Several mature mRNAs of Trypanosoma brucei were previously shown to have a common 5' terminal sequence of 35 nucleotides (nt) encoded by a separate mini-exon. To verify whether all trypanosome mRNAs contain this mini-exon sequence at their 5' end, we have tested oligodeoxynucleotides complementary to different parts of the 35 nt leader sequence for their ability to inhibit translation of total trypanosome mRNA. All oligomers tested inhibited translation of trypanosome mRNAs in a wheat germ extract. They had no effect on translation of Brome mosaic virus mRNA and of a trypanosome mRNA for phosphoglycerate kinase modified to remove the mini-exon sequence. Three different 12mers inhibited translation 35-60%; both the 22- and 34mer inhibited translation 95-100%. Incorporation of amino acids decreased proportionally in all protein bands detected in high resolution polyacrylamide gels. Our results show that all trypanosome mRNAs that yield a product detectable in gel contain a mini-exon sequence. We infer that most, if not all, trypanosome mRNAs contain a 5' terminal mini-exon sequence acquired by discontinuous synthesis.     

Identification of a novel Y branch structure as an intermediate in trypanosome mRNA processing: evidence for trans splicing

We present evidence that addition of the 35 nucleotide spliced leader (SL) to the 5' end of T. brucei mRNAs occurs via trans RNA splicing. A 100 nucleotide fragment of the 135 base SL RNA (100-mer) is revealed by S1 nuclease analysis of total and poly(A)+ RNA. This 100-mer is not detected by Northern hybridization analysis, indicating that it does not exist free in the cell. The 5' end of the 100-mer maps precisely to the conserved splice junction sequence of the SL RNA. Purified debranching enzyme releases this 100-mer RNA as a free, 100 nucleotide species. This indicates that the 100-mer is covalently linked to poly(A)+ RNA by a 2'-5' phosphodiester bond, that the branched intermediate has a discontinuous intron or Y structure (rather than a lariat), which is expected of a trans-spliced mRNA, and that the SL RNA is indeed the donor of the SL sequence to trypanosome mRNAs.     

Discordant expression and variable numbers of neighboring GGA- and GAA-rich triplet repeats in the 3' untranslated regions of two groups of messenger RNAs encoded by the rat polymeric immunoglobulin receptor gene.

An unusual S1-nuclease sensitive microsatellite (STMS) has been found in the single copy, rat polymeric immunoglobulin receptor gene (PIGR) terminal exon. In Fisher rats, elements within or beyond the STMS are expressed variably in the 3' untranslated regions (3'UTRs) of two 'Groups' of PIGR-encoded hepatic mRNAs (pIg-R) during liver regeneration. STMS elements include neighboring constant regions (a 60-bp d[GA]-rich tract with a chi-like octamer, followed by 15 tandem d[GGA] repeats) that merge directly with 36 or 39 tandem d[GAA] repeats (Fisher or Wistar strains, respectively) interrupted by d[AA] between their 5th-6th repeat units. The Wistar STMS is flanked upstream by two regions of nearly contiguous d[CA] or d[CT] repeats in the 3' end of intron 8; and downstream, by a 283 bp 'unit' containing several inversions at its 5' end, and two polyadenylation signals at its 3' end. The 283 nt unit is expressed in Group 1 pIg-R mRNAs; but it is absent in the Group 2 family so that their GAA repeats merge with their poly A tails. In contrast to genomic sequence, GGA triplet repeats are amplified (n > or = 24-26), whereas GAA triplet repeats are truncated variably (n < or = 9-37) and expressed uninterruptedly in both mRNA Groups. These results suggest that 3' end processing of the rat PIGR gene may involve misalignment, slippage and premature termination of RNA polymerase II. The function of this unusual processing and possible roles of chi-like octamers in quiescent or extrahepatic tissues are discussed.     

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.     

Tandemly arranged variant 5S ribosomal RNA genes in the yeast Saccharomyces cerevisiae.

Most of the ribosomal RNA genes of the yeast Saccharomyces cerevisiae are about 9 kilobases (kb) in size and encode both the 35S rRNA (processed to produce the 25S, 18S, and 5.8S species) and 5S rRNA. These genes are arranged in a single tandem array of 100 repeats. Below, we present evidence that at the centromere-distal end of this array is a tandem arrangement of a different type of rRNA gene. Each of these repeats is 3.6 kb in length and encodes a single 5S rRNA. The coding sequence of this gene is different from that of the "normal" 5S gene in three positions located at the 3' end of the gene.     

Glutamate tRNA genes are adjacent to 5S RNA genes in Drosophila and reveal a conserved upstream sequence (the ACT-TA box).

In Drosophila melanogaster at least six transfer RNA genes are located adjacent to the 3' end of the 5S RNA gene cluster. Three of these have been sequenced and identified as coding for glutamate tRNA4. In the chromosome they are arranged as tandem repeats on the same DNA strand and transcribed in the same direction as is 5S DNA, towards the centromere. We have also identified a sequence, the ACT-TA box, that is highly conserved among the polymerase III transcribed genes. Usually the sequence is located at 37 +/- 8 base pairs upstream from the first nucleotide of the structural gene. A similar sequence is also observed upstream of yeast and silkworm tRNA genes and the mitochondrial tRNA genes of mouse and humans.     

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.     

Comparison of Rous sarcoma virus RNA processing in chicken and mouse fibroblasts: evidence for double-spliced RNA in nonpermissive mouse cells.

Rous sarcoma virus, an avian retrovirus, transforms but does not replicate in mammalian cells. To determine to what extent differences in RNA splicing might contribute to this lack of productive infection, cloned proviral DNA derived from the Prague A strain of Rous sarcoma virus was transfected into mouse NIH 3T3 cells, and the viral RNA was compared by RNase protection with viral RNA from transfected chicken embryo fibroblasts by using a tandem antisense riboprobe spanning the three major splice sites. The levels of viral RNA in NIH 3T3 cells compared with those in chicken embryo fibroblasts were lower, but the RNA was spliced at increased efficiency. The difference in the ratio of unspliced to spliced RNA levels was not due to the increased lability of unspliced RNA in NIH 3T3 cells. Although chicken embryo fibroblasts contained equal levels of src and env mRNAs, spliced viral mRNAs in NIH 3T3 cells were almost exclusively src. In NIH 3T3 cells the env mRNA was further processed by using a cryptic 5' splice site located within the env coding sequences and the normal src 3' splice site to form a double-spliced mRNA. This mRNA was identical to the src mRNA, except that a 159-nucleotide sequence from the 5' end of the env gene was inserted at the src splice junction. Smaller amounts of single-spliced RNA were also present in which only the region between the cryptic 5' and src 3' splice sites was spliced out. The aberrant processing of the viral env mRNA in NIH 3T3 cells may in part explain the nonpermissiveness of these cells to productive Rous sarcoma virus infection.     

Genes encoding proteins with homologous contiguous repeat sequences are highly expressed in the serous cells of the rat submandibular gland

An abundant class of secreted salivary polypeptides is characterized by the presence of identical and contiguous repeats of amino acid sequences within the polypeptide chains, and includes the proline-rich proteins. We discovered a new family of contiguous repeat polypeptides (CRPs) that is related to the proline-rich proteins but contains little proline. Analysis of salivary mRNAs and liver DNA by molecular cloning, DNA sequence determinations, and Northern and Southern blot hybridization revealed several closely related CRP mRNAs and at least 10 CRP-related genes. We further analyzed two CRP mRNAs of 850 and 920 nucleotides and the gene encoding the larger CRP mRNA. The two mRNAs contain the same 69-base repeats in their coding regions and are identical in their 5'- and 3'-untranslated tracts. However, they differ in the number of contiguous repeats (four versus five) and a segment at the 3' end of the coding region which encodes closely related but unique COOH termini of the CRPs. These structural features suggest a recent gene conversion. The CRP gene analyzed is divided into three exons that encode (i) 5'-untranslated tract and signal sequence, (ii) secreted polypeptide, and (iii) 3'-untranslated tract, respectively. CRP mRNA contains two open reading frames. The longer open reading frame encodes a CRP precursor with a signal sequence of 17 amino acids, four to five contiguous repeats of 23 amino acids, and a variable COOH region that begins with two segments related to the contiguous repeats. Immunochemical analysis of salivary gland slices with antisera raised against peptides corresponding to two regions of the larger open reading frame revealed intense staining only of the serous cells of the submandibular glands. 35S-Labeled oligonucleotides complementary to CRP mRNA specifically hybridized to the same cells.