Dicer-like (DCL) proteins in plants

Dicer and Dicer-like (DCL) proteins are key components in small RNA biogenesis. DCLs form a small protein family in plants whose diversification time dates to the emergence of mosses (Physcomitrella patens). DCLs are ubiquitously but not evenly expressed in tissues, at different developmental stages, and in response to environmental stresses. In Arabidopsis, AtDCL1, AtDCL2, and AtDCL4 exhibit similar expression pattern during the leaf or stem development, which is distinguished from AtDCL3. However, distinct expression profiles for all DCLs are found during the development of reproductive organs flower and seed. The grape VvDCL1 and VvDCL3 may act sequentially to face the fungi challenge. Overall, the responses of DCLs to drought, cold, and salt are quite different, indicating that plants might have specialized regulatory mechanism in response to different abiotic stresses. Further analysis of the promoter regions reveals a few of cis-elements that are hormone- and stress-responsive and developmental-related. However, gain and loss of cis-elements are frequent during evolution, and not only paralogous but also orthologous DCLs have dissimilar cis-element organization. In addition to cis-elements, AtDCL1 is probably regulated by both ath-miR162 and ath-miR414. Posterior analysis has identified some critical amino acid sites that are responsible for functional divergence between DCL family members. These findings provide new insights into understanding DCL protein functions. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Escherichia coli endoribonuclease RNase E: autoregulation of expression and site-specific cleavage of mRNA

Mutations in the Escherichia coli rne (ams) gene have a general effect on the rate of mRNA decay in vivo. Using antibodies we have shown that the product of the rne gene is a polypeptide of relative mobility 180kDa. However, proteolytic fragments as small as 70kDa, which can arise during purification, also exhibit RNase E activity, in vitro studies demonstrate that the rne gene product, RNase E, is an endoribonuclease that cleaves mRNA at specific sites. RNase E cleaves rne mRNA and autoregulates the expression of the rne gene. In addition we demonstrate RNase E-dependent endonucleolytic cleavage of ompA mRNA, at a site known to be rate-determining for degradation and reported to be cieaved by RNase K. Our data are consistent with RNase K being a proteolytic fragment of RNase E.     

Expression of lexA targeted ribozyme in Escherichia coli BL-21 (DE3) cells

Coding sequences for a hammerhead ribozyme designed to cleave lexA mRNA in a targeted manner was cloned under phage T7 promoter and expressed in E. coli strain BL-21 (DE3) expressing T7 RNA polymerase under the control of IPTG-inducible lac UV-5 promoter. Ribozyme expression in vivo was demonstrated by RNase protection assay. Also, total RNA extracted from these transformed cells following induction by IPTG, displays site-specific cleavage of labeled lexA RNA in an In vitro reaction. The result demonstrates the active ribozyme in extracts of cell transformed with a recombinant cassette and goes beyond the earlier demonstration of the stability of In vitro synthesized ribozyme in cell extracts. The observed rise in lexA mRNA rules out any role for protease activity or resulting fragments of lexA protein in de-repression of RNA. (Mol Cell Biochem 271: 197–203, 2005)     

The ribosome binding site of a mini‐ORF protects a T3SS mRNA from degradation by RNase E

Enterohaemorrhagic Escherichia coli harbours a pathogenicity island encoding a type 3 secretion system used to translocate effector proteins into the cytosol of intestinal epithelial cells and subvert their function. The structural proteins of the translocon are encoded in a major espADB mRNA processed from a precursor. The translocon mRNA should be highly susceptible to RNase E cleavage because of its AU‐rich leader region and monophosphorylated 5′‐terminus, yet it manages to avoid rapid degradation. Here, we report that the espADB leader region contains a strong Shine–Dalgarno element (SD2) and a translatable mini‐ORF of six codons. Disruption of SD2 so as to weaken ribosome binding significantly reduces the concentration and stability of esp mRNA, whereas codon substitutions that impair translation of the mini‐ORF have no such effect. These findings suggest that occupancy of SD2 by ribosomes, but not mini‐ORF translation, helps to protect espADB mRNA from degradation, likely by hindering RNase E access to the AU‐rich leader region.     

Functional Conservation of RNase III-like Enzymes: Studies on a Vibrio vulnificus Ortholog of Escherichia coli RNase III

Bacterial ribonuclease III (RNase III) belongs to the RNase III enzyme family, which plays a pivotal role in controlling mRNA stability and RNA processing in both prokaryotes and eukaryotes. In the Vibrio vulnificus genome, one open reading frame encodes a protein homologous to E. coli RNase III, designated Vv-RNase III, which has 77.9 % amino acid identity to E. coli RNase III. Here, we report that Vv-RNase III has the same cleavage specificity as E. coli RNase III in vivo and in vitro. Expressing Vv-RNase III in E. coli cells deleted for the RNase III gene (rnc) restored normal rRNA processing and, consequently, growth rates of these cells comparable to wild-type cells. In vitro cleavage assays further showed that Vv-RNase III has the same cleavage activity and specificity as E. coli RNase III on RNase III-targeted sequences of corA and mltD mRNA. Our findings suggest that RNase III-like proteins have conserved cleavage specificity across bacterial species.     

Determinants in the rpsT mRNAs recognized by the 5′‐sensor domain of RNase E

RNase E plays a central role in processing virtually all classes of cellular RNA in many bacterial species. A characteristic feature of RNase E and its paralogue RNase G, as well as several other unrelated ribonucleases, is their preference for 5′‐monophosphorylated substrates. The basis for this property has been explored in vitro. At limiting substrate, cleavage of the rpsT mRNA by RNase E (residues 1–529) is inefficient, requiring excess enzyme. The rpsT mRNA is cleaved sequentially in a 5′ to 3′ direction, with the initial cleavage(s) at positions 116/117 or 190/191 being largely driven by direct entry, independent of the 5′‐terminus or the 5′‐sensor domain of RNase E. Generation of the 147 nt 3′‐limit product requires sequential cleavages that generate 5′‐monophosphorylated termini on intermediates, and the 5′‐sensor domain of RNase E. These requirements can be bypassed with limiting enzyme by deleting a stem‐loop structure adjacent to the site of the major, most distal cleavage. Alternatively, this specific cleavage can be activated substantially by a 5′‐phosphorylated oligonucleotide annealed 5′ to the cleavage site. This finding suggests that monophosphorylated small RNAs may destabilize their mRNA targets by recruiting the 5‐sensor domain of RNase E ‘in trans’.     

Structure and regulation of the Salmonella typhimurium rnc-era-recO operon

The Escherichia coli rnc-era-recO operon encodes ribonuclease III (RNase III; a dsRNA endonuclease involved in rRNA and mRNA processing and decay), Era (an essential G-protein of unknown function) and RecO (involved in the RecF homologous recombination pathway). Expression of the rnc and era genes is negatively autoregulated: RNase III cleaves the rncO ‘operator’ in the untranslated leader, destabilizing the operon mRNA. As part of a larger effort to understand RNase III and Era structure and function, we characterized rnc operon structure, function and regulation in the closely related bacterium Salmonella typhimurium. Construction of a S typhimurium strain conditionally defective for RNase III and Era expression showed that Era is essential for cell growth. This mutant strain also enabled selection of recombinant clones containing the intact S typhimurium rnc-era-recO operon, whose nucleotide sequence, predicted protein sequence, and predicted rncO RNA secondary structure were all highly conserved with those of E coli. Furthermore, genetic and biochemical analysis revealed that S typhimurium rnc gene expression is negatively autoregulated by a mechanism very similar or identical to that in E coli, and that the cleavage specificities of RNase III_S.t. and RNase III_E.c. are indistinguishable with regard to rncO cleavage and S typhimurium 23S rRNA fragmentation in vivo.     

The response regulator RpaB binds the high light regulatory 1 sequence upstream of the high-light-inducible <Emphasis Type="Italic">hliB</Emphasis> gene from the cyanobacterium <Emphasis Type="Italic">Synechocystis</Emphasis> PCC 6803

Cyanobacteria, like other photosynthetic organisms, respond to the potentially damaging effects of high-intensity light by regulating the expression of a variety of stress-responsive genes through regulatory mechanisms that remain poorly understood. The high light regulatory 1 (HLR1) sequence can be found upstream of many genes regulated by high-light (HL) stress in cyanobacteria. In this study, we identify the factor that binds the HLR1 upstream of the HL-inducible hliB gene in the cyanobacterium Synechocystis PCC 6803 as the RpaB (Slr0947) response regulator.