mRNA Processing Events Research Articles

RNA polymerase II elongation control.

Regulation of the elongation phase of transcription by RNA polymerase II (Pol II) is utilized extensively to generate the pattern of mRNAs needed to specify cell types and to respond to environmental changes. After Pol II initiates, negative elongation factors cause it to pause in a promoter proximal position. These polymerases are poised to respond to the positive transcription elongation factor P-TEFb, and then enter productive elongation only under the appropriate set of signals to generate full-length properly processed mRNAs. Recent global analyses of Pol II and elongation factors, mechanisms that regulate P-TEFb involving the 7SK small nuclear ribonucleoprotein (snRNP), factors that control both the negative and positive elongation properties of Pol II, and the mRNA processing events that are coupled with elongation are discussed.

MRNA capping enzyme is recruited to the transcription complex by phosphorylation of the RNA polymerase II carboxy-terminal domain.

Capping of mRNA occurs shortly after transcription initiation, preceding other mRNA processing events such as mRNA splicing and polyadenylation. To determine the mechanism of coupling between transcription and capping, we tested for a physical interaction between capping enzyme and the transcription machinery. Capping enzyme is not stably associated with basal transcription factors or the RNA polymerase II (Pol II) holoenzyme. However, capping enzyme can directly and specifically interact with the phosphorylated form of the RNA polymerase carboxy-terminal domain (CTD). This association occurs in the context of the transcription initiation complex and is blocked by the CTD-kinase inhibitor H8. Furthermore, conditional truncation mutants of the Pol II CTD are lethal when combined with a capping enzyme mutant. Our results provide in vitro and in vivo evidence that capping enzyme is recruited to the transcription complex via phosphorylation of the RNA polymerase CTD.

Mutations affecting mRNA processing and fimbrial biogenesis in the Escherichia coli pap operon

The Escherichia coli pap genetic determinant includes 11 genes and encodes expression of Pap pili on the bacterial surface. An RNase E-dependent mRNA-processing event in the intercistronic papB-papA region results in the accumulation of a papA-gene-specific mRNA in considerable excess of the primary papB-papA mRNA transcription product. We have introduced mutations in the intercistronic region and studied the effect in vivo of these mutations on the processing event, PapA protein expression, and the biogenesis of fimbriae on the bacterial surface. Our studies establish that mRNA processing is an important event in the mechanism resulting in differential gene expression of the major pap operon. The deletion of sequences corresponding to the major cleavage site abolished processing, reduced expression of PapA protein, and resulted in "crew-cut" bacteria with short fimbrial structures on the bacterial surface. Only a limited part of the intercistronic region appeared to be required as the recognized target for the processing to occur. Upstream sequences to a position within 10 nucleotides of the major RNase E-dependent cleavage site could be deleted without any detectable effect on papB-papA mRNA processing, PapA protein expression, or fimbria formation. Substitution mutations of specific bases at the cleavage site by site-directed mutagenesis showed that there were alternative positions at which cleavage could be enhanced, and tests with an in vitro processing assay showed that such cleavages were also RNase E dependent. Our findings are discussed in relation to other fimbrial operons and other known targets of the RNase E endoribonuclease.

Differential decay of a polycistronic Escherichia coli transcript is initiated by RNaseE‐dependent endonucleolytic processing

Differential expression of the genes expressing Pap pili in Escherichia coli was suggested to involve mRNAs with different stabilities. As the result of a post-transcriptional processing event, a papA gene-specific mRNA product (mRNA-A) accumulates in large excess relative to the primary mRNA-BA transcript. Our results show that the processed product, mRNA-A, is a translationally active molecule and that it is generated from the mRNA-BA precursor by an RNaseE-dependent mechanism. The processing did not occur under non-permissive conditions in an E. coli rne mutant strain with a temperature-sensitive RNaseE. The endonuclease RNaseE was previously described as being chiefly involved in the processing of the 9S precursor of 5S rRNA. A comparison of nucleotide sequences of mRNA-BA and three other RNAs processed by RNAseE revealed a conserved motif around the cleavage sites. Mutations abolishing the activity of either of two other endoribonucleases, RNaseIII and RNaseP, did not affect the pap mRNA processing event. However, a conditional mutation in the ams locus, causing altered stability of bulk mRNA in E. coli, led to reduced pap mRNA processing in a manner similar to the effect caused by RNaseE deficiency. Our findings are consistent with the idea that ams is related/allelic to rne. Absence of the processing event in the RNaseE mutant (rne-3071) strain led to a four-fold stabilization of the mRNA-BA primary transcript. We conclude that the RNaseE-dependent processing event is the rate-limiting step in the decay of the papB-coding part of the primary transcript and in the production of the stable mRNA-A product.(ABSTRACT TRUNCATED AT 250 WORDS)

2'-O-methyl, 2'-O-ethyl oligoribonucleotides and phosphorothioate oligodeoxyribonucleotides as inhibitors of the in vitro U7 snRNP-dependent mRNA processing event

We describe the synthesis of 2'-O-methyl, 2'-O-ethyl oligoribonucleotides and phosphorothioate oligodeoxyribonucleotides and demonstrate their utility as inhibitors of the in vitro U7 snRNP-dependent mRNA processing event. These 2'-O-modified compounds were designed to possess the binding affinity of an RNA molecule towards a complementary RNA target with an enhanced stability against nucleases. The 2'-O-methyl and 2'-O-ethyl antisense compounds function as potent inhibitors of the reaction at 1-10 nM, approximately 5-fold more effective than a natural antisense RNA molecule and requiring an approximate 5-fold excess over the target RNA for 80% inhibition of the processing reaction.

Characterization of the apolipoprotein B mRNA editing enzyme: no similarity to the proposed mechanism of RNA editing in kinetoplastid protozoa.

Intestinal apolipoprotein B mRNA is edited at nucleotide 6666 by a C to U transition resulting in a translational stop codon. The enzymatic properties of the editing activity were characterised in vitro using rat enterocyte cytosolic extract. The editing activity has no nucleotide or ion cofactor requirement. It shows substrate saturation with an apparent Km for the RNA substrate of 2.2 nM. The editing enzyme requires no lag period prior to catalysis, and does not assemble into a higher order complex on the RNA substrate. In crude cytosolic extract editing activity is completely abolished by treatment with micrococcal nuclease or RNAse A. Partially purified editing enzyme is no longer sensitive to nucleases, but is inhibited in a dose dependent manner by nuclease inactivated crude extract. The buoyant density of partially purified editing enzyme is 1.3 g/ml, that of pure protein. Therefore, the apolipoprotein B mRNA editing activity consists of a well defined enzyme with no RNA component. The nuclease sensitivity in crude cytosolic extract is explained by the generation of inhibitors for the editing enzyme. The editing of apo B mRNA has little similarity to complex mRNA processing events such as splicing and unlike editing in kinetoplastid protozoa does not utilise guide RNAs.

Post-transcriptional regulation of thymidine kinase gene expression during monocytic differentiation of HL60 promyelocytes

The regulatory mechanism of human thymidine kinase (TK) gene expression was investigated in HL-60 promyelocytes during induction of monocytic differentiation with 12-O-tetradecanoyl phorbol-13-acetate (TPA). The steady-state levels of TK mRNA diminished gradually as cells were treated with TPA. The nuclear run-on experiments were performed and revealed that TPA treatment did not change TK gene activity in HL-60 cells. These findings suggested that the expression of TK mRNA was controlled by a post-transcriptional mechanism. The half-life of mature TK mRNA transcript was found to be more than 8 hours in both proliferating and differentiated HL-60 cells, which indicated that the stability of mature TK mRNA does not play a role in regulating TK gene expression. Analysis of poly(A-) TK mRNAs showed the high molecular weight precursors of TK mRNA which appeared in proliferating cells were not detectable in TPA-treated cells. This finding suggested that the TK mRNA processing event is implicated in the regulation of human TK gene expression in HL-60 cells during monocytic terminal differentiation.

Inhibition of methylation at two internal N6-methyladenosine sites caused by GAC to GAU mutations.

We previously have mapped N6-methyladenosine (m6A) sites within the genomic RNA of Rous sarcoma virus (RSV). The results of that study and of experiments using inhibitors of methylation suggest that m6A might be involved in mRNA processing events. We describe an approach for directly analyzing the function of m6A in RNA and for studying the sequence specificity of the m6A methylase. Two sites of methylation in RSV (nucleotides 7414 and 7424) were altered by oligonucleotide-directed mutagenesis. The highly conserved GAC consensus sequence at those sites was changed to GAU. The new sequences were no longer methylated in the RSV genomic RNA; the GAC sequence was required for efficient base modification at those two adenosines. The altered m6A pattern did not affect viral RNA processing or the viral life cycle within infected cells.

Initiation, attenuation and RNase III processing of transcripts from the Escherichia coli operon encoding ribosomal protein S15 and polynucleotide phosphorylase

The rpsO gene of Escherichia coli, which encodes ribosomal protein S15 is located at 69 minutes on the chromosome. It is adjacent to the pnp gene, which encodes polynucleotide phosphorylase. The two genes are separated by 249 nucleotides and are transcribed in the same direction. We report here in vivo S 1 nuclease mapping and in vitro transcription experiments that demonstrate that rpsO and pnp are cotranscribed from a promoter P 1, located 108 nucleotides upstream from rpsO, and that another promoter P 2, located between the two genes 158 nucleotides upstream from pnp, also directs the transcription of pnp. Transcription from P 1 can either terminate at the terminator t 1 identified in vivo and in vitro, 18 nucleotides downstream from rpsO, or transcribe through t 1 and into pnp. Comparison of the transcripts synthesized in wild-type and RNase III-deficient strains of E. coli shows that all the P 1 readthrough transcripts and P 2 transcripts are cleaved by RNase III. Two specific cuts are made by RNase III in a double-stranded structure about 100 nucleotides upstream from rpsO. We also found that some transcripts of this operon start 47 nucleotides downstream from rpsO, in the region of t 1. No promoter has been identified in this region. This mRNA is attributed to an endonucleolytic cleavage of the polycistronic transcripts and the location of the cut is named M. The order of the transcription signals and of the maturation sites in relation to rpsO and pnp can be summarized as follows: P 1, rpsO, t 1, M, P 2, RNase III-processing sites, pnp. The possible roles of mRNA processing events in the expression of rpsO-pnp operon are discussed.

MRNA processing inEscherichia coli: an activity encoded by the host processes bacteriophage f1 mRNAs

To examine the regions of the male-specific filamentous bacteriophage f1 genome that include signals for mRNA processing, the 5' endpoints of the major in vivo phage mRNAs have been located in the f1 DNA sequence by S1 nuclease mapping. The 5' ends of the purified mRNAs and additional phage-specific RNAs transiently visible early after infection occur in clusters of T-rich residues within genes that code for three phage proteins. When a 270-nucleotide region encompassing the 5' endpoints of three processed RNAs is transcribed as part of the bacteriophage lambda N mRNA in uninfected female cells, RNA 5' ends identical to ends of the three f1 RNAs are generated from the lambda-f1 precursor. This finding indicates that the mRNA processing activity is encoded by the bacterial host, and that its recognition sites are present in the local regions near the 5' ends which result from RNA cleavage. Several characteristics of f1 mRNA processing events have implications for the differential regulation of adjacent phage genes constrained in the same transcription unit, and may be representative of similar processing events occurring in the bacterial cell.

Rabbit heavy chain haplotypes ? Allotypic determinants expressed byVH-CH recombinants

This report summarizes our current understanding of the heavy chain haplotypes found in our laboratories' rabbits. Independently derived data from several laboratories have been synthesizes into a consistent picture of the linked inheritance of allotypic markers found on the different heavy chain classes and subclasses of rabbit immunoglobulins in pedigreed rabbits, including the families of three apparent VH-CH recombinants. In one recombinant, the entire group of CH markers (C mu, C gamma, and C alpha) recombined with the set of VH. Although in the other two recombinants all CH markers may also have recombined as a group, in one of these only IgG and IgA CH genes were informative; in the other recombinant, only the IgG allotypes were informative. Some allotypic determinants found on IgM molecules ("conformational") appear only when a specific variable region allotype (VHa) is combined with a specific mu constant region allotype (C mu). New combinations of VHa and C mu allotypes were generated in two of the genetic recombinants and led to new "conformational" determinants. The gains and losses observed lend support to the hypothesis that the determinants result from conformations generated by the combination of allotype-specific VH and C mu protein sequences. Conceivably, DNA events that join VH to diversity (D)- and joining (J)-coding sequences or mRNA processing events that splice J to C mu could be involved in generating the sequences that form allotype-specific determinants.