Multiple products from microRNA transcripts

A single transcript sometimes codes for more than one product. In bacteria, and in a few exceptional animal lineages, many genes are organized into operons: clusters of open reading frames that are transcribed together in a single polycistronic transcript. However, polycistronic transcripts are rare in eukaryotes. One notable exception is that of miRNAs (microRNAs), small RNAs that regulate gene expression at the post-transcriptional level. The primary transcripts of miRNAs commonly produce more than one functional product, by at least three different mechanisms. miRNAs are often produced from polycistronic transcripts together with other miRNA precursors. Also, miRNAs frequently derive from protein-coding gene introns. Finally, each miRNA precursor can produce two mature miRNA products. We argue, in the present review, that miRNAs are frequently hosted in transcripts coding for multiple products because new miRNA precursor sequences that arise by chance in transcribed regions are more likely to become functional miRNAs during evolution.

Autoregulation of AbsB (RNase III) Expression in Streptomyces coelicolor by Endoribonucleolytic Cleavage of absB Operon Transcripts

ABSTRACT The Streptomyces coelicolor absB gene encodes an RNase III family endoribonuclease and is normally essential for antibiotic biosynthesis. Here we report that AbsB controls its own expression by sequentially and site specifically cleaving stem-loop segments of its polycistronic transcript. Our results demonstrate a ribonucleolytic regulatory role for AbsB in vivo.

Pneumocystis Encodes a Functional S-Adenosylmethionine Synthetase Gene

ABSTRACT S-Adenosylmethionine (AdoMet) synthetase (EC 2.5.1.6) is the enzyme that catalyzes the synthesis of AdoMet, a molecule important for all cellular organisms. We have cloned and characterized an AdoMet synthetase gene (sam1) from Pneumocystis spp. This gene was transcribed primarily as an ∼1.3-kb mRNA which encodes a protein containing 381 amino acids in P. carinii or P. murina and 382 amino acids in P. jirovecii. sam1 was also transcribed as part of an apparent polycistronic transcript of ∼5.6 kb, together with a putative chromatin remodeling protein homologous to Saccharomyces cerevisiae, CHD1. Recombinant Sam1, when expressed in Escherichia coli, showed functional enzyme activity. Immunoprecipitation and confocal immunofluorescence analysis using an antipeptide antibody showed that this enzyme is expressed in P. murina. Thus, Pneumocystis, like other organisms, can synthesize its own AdoMet and may not depend on its host for the supply of this important molecule.

Transcription and Analysis of Polymorphism in a Cluster of Genes Encoding Surface-Associated Proteins of Clostridium difficile

ABSTRACT Recent investigations of the Clostridium difficile genome have revealed the presence of a cluster of 17 genes, 11 of which encode proteins with similar two-domain structures, likely to be surface-anchored proteins. Two of these genes have been proven to encode proteins involved in cell adherence: slpA encodes the precursor of the two proteins of the S-layer, P36 and P47, whereas cwp66 encodes the Cwp66 adhesin. To gain further insight into the function of this cluster, we further focused on slpA, cwp66, and cwp84, the latter of which encodes a putative surface-associated protein with homology to numerous cysteine proteases. It displayed nonspecific proteolytic activity when expressed as a recombinant protein in Escherichia coli. Polymorphism of cwp66 and cwp84 genes was analyzed in 28 strains, and transcriptional organization of the three genes was explored by Northern blots. The slpA gene is strongly transcribed during the entire growth phase as a bicistronic transcript; cwp66 is transcribed only in the early exponential growth phase as a polycistronic transcript encompassing the two contiguous genes upstream. The putative proteins encoded by the cotranscribed genes have no significant homology with known proteins but may have a role in adherence. No correlation could be established between sequence patterns of Cwp66 and Cwp84 and virulence of the strains. The cwp84 gene is strongly transcribed as a monocistronic message. This feature, together with the highly conserved sequence pattern of cwp84, suggests a significant role in the physiopathology of C. difficile for the Cwp84 protease, potentially in the maturation of surface-associated adhesins encoded by the gene cluster.

Early Cephamycin Biosynthetic Genes Are Expressed from a Polycistronic Transcript in Streptomyces clavuligerus

ABSTRACT A polycistronic transcript that is initiated at the latpromoter has been implicated in the expression of the genes involved in early steps of cephamycin C biosynthesis in Streptomyces clavuligerus. pcbC is also expressed as a monocistronic transcript from its own promoter. However, an alternative interpretation involving expression via three separate yet interdependent transcripts has also been proposed. To distinguish between these possibilities, mutants lacking the latpromoter and containing a transcription terminator within thelat gene (Δlat::tsr/term mutants) were created. This mutation eliminated the production of lysine-ɛ-aminotransferase (the lat gene product) but also affected the expression of downstream genes, indicating an operon arrangement. Production of δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine synthetase (ACVS) (the pcbAB gene product) was eliminated in Δlat::tsr/term mutants, while production of isopenicillin N synthase (IPNS) (the pcbCgene product) was greatly reduced. The provision of α-aminoadipate to the Δlat::tsr/term mutants, either via exogenous feeding or via lat gene complementation, did not restore production of ACVS or IPNS. Analysis of RNA isolated from the Δlat::tsr/term mutants confirmed that the polycistronic transcript was absent but also indicated that monocistronic pcbC transcript levels were greatly decreased. In contrast, Δlat mutants created by in-frame internal deletion of lat maintained the polycistronic transcript and allowed production of wild-type levels of both ACVS and IPNS.

Seven Novel Methylation Guide Small Nucleolar RNAs Are Processed from a Common Polycistronic Transcript by Rat1p and RNase III in Yeast

ABSTRACT Through a computer search of the genome of the yeastSaccharomyces cerevisiae, the coding sequences of seven different box C/D antisense small nucleolar RNAs (snoRNAs) with the structural hallmarks of guides for rRNA ribose methylation have been detected clustered over a 1.4-kb tract in an inter-open reading frame region of chromosome XIII. The corresponding snoRNAs have been positively identified in yeast cells. Disruption of the nonessential snoRNA gene cluster specifically suppressed the seven cognate rRNA ribose methylations but did not result in any growth delay under the conditions of yeast culture tested. The seven snoRNAs are processed from a common polycistronic transcript synthesized from an independent promoter, similar to some plant snoRNAs but in marked contrast with their vertebrate functional homologues processed from pre-mRNA introns containing a single snoRNA. Processing of the polycistronic precursor requires nucleases also involved in rRNA processing, i.e., Rnt1p and Rat1p. After disruption of the RNT1 gene, the yeast ortholog of bacterial RNase III, production of the seven mature snoRNAs was abolished, while the polycistronic snoRNA precursor accumulated. In cells lacking functional Rat1p, an exonuclease involved in the processing of both pre-rRNA and intron-encoded snoRNAs, several processing intermediates of the polycistronic precursor accumulated. This allowed for the mapping in the precursor of the presumptive Rnt1p endonucleolytic cuts which provide entry sites for subsequent exonucleolytic trimming of the pre-snoRNAs. In line with known properties of double-stranded RNA-specific RNase III, pairs of Rnt1p cuts map next to each other on opposite strands of long double-helical stems in the secondary structure predicted for the polycistronic snoRNA precursor.