Small, Smaller, Smallest: Minimal Structural Requirements for a Fully Functional Box C/D Modification Guide RNA

Site-specific 2’-O-ribose methylation is an abundant post-transcriptional modification mediated by small non-coding nuclear RNAs known as box C/D modification guide RNAs. The minimal structural requirements for these guide RNAs to function in higher eukaryotes are still unclear. To address this question, we generated a series of mutant variants of Drosophila box C/D scaRNA:MeU2-C28 and tested their modification guide activities in the Xenopus oocyte system. Our data suggest that box C/D guide RNA function requires either a terminal or an internal consensus kink-turn structure. We identified the minimal functional box C/D guide RNA. It consists of a single-domain molecule with (i) a terminal stem with a consensus kink-turn domain, (ii) one box C and box D connected by a 14-nucleotide antisense element and (iii) a one-nucleotide spacer between the box C and the antisense element. In this single domain RNA, the sequence of the spacer is more important than its length. We suggest that the secondary structure of box C/D RNAs, essential for guide RNA function, is more complex than generally supposed. At the same time, the expression of functional extremely short single-domain box C/D RNAs is possible in higher eukaryotes.

Limited Role for the DsrA and RprA Regulatory RNAs in rpoS Regulation in Salmonella enterica

ABSTRACT RpoS, the sigma factor of enteric bacteria that responds to stress and stationary phase, is subject to complex regulation acting at multiple levels, including transcription, translation, and proteolysis. Increased translation of rpoS mRNA during growth at low temperature, after osmotic challenge, or with a constitutively activated Rcs phosphorelay depends on two trans-acting small regulatory RNAs (sRNAs) in Escherichia coli. The DsrA and RprA sRNAs are both highly conserved in Salmonella enterica, as is their target, an inhibitory antisense element within the rpoS untranslated leader. Analysis of dsrA and rprA deletion mutants indicates that while the increased translation of RpoS in response to osmotic challenge is conserved in S. enterica, dependence on these two sRNA regulators is much reduced. Furthermore, low-temperature growth or constitutive RcsC activation had only modest effects on RpoS expression, and these increases were, respectively, independent of dsrA or rprA function. This lack of conservation of sRNA function suggests surprising flexibility in RpoS regulation.

Role of ppGpp in rpoS Stationary-Phase Regulation in Escherichia coli

ABSTRACT The bacterial sigma factor RpoS is strongly induced under a variety of stress conditions and during growth into stationary phase. Here, we used rpoS-lac fusions in Escherichia coli to investigate control acting at the level of RpoS synthesis, which is especially evident when cells approach stationary phase in rich medium. Previous work has shown that the small molecule ppGpp is required for normal levels of RpoS in stationary phase. Despite the attraction of a model in which the ppGpp level controls stationary-phase induction of RpoS, careful measurement of rpoS-lac expression in a mutant lacking ppGpp showed similar effects during both exponential growth and stationary phase; the main effect of ppGpp was on basal expression. In addition, a modest regulatory defect was associated with the mutant lacking ppGpp, delaying the time at which full expression was achieved by 2 to 3 h. Deletion analysis showed that the defect in basal expression was distributed over several sequence elements, while the regulatory defect mapped to the region upstream of the rpoS ribosome-binding site (RBS) that contains a cis-acting antisense element. A number of other genes that have been suggested as regulators of rpoS were tested, including dksA, dsrA, barA, ppkx, and hfq. With the exception of the dksA mutant, which had a modest defect in Luria-Bertani medium, none of these mutants was defective for rpoS stationary-phase induction. Even a short rpoS segment starting at 24 nucleotides upstream of the AUG initiation codon was sufficient to confer substantial stationary-phase regulation, which was mainly posttranscriptional. The effect of RBS-proximal sequence was independent of all known trans-acting factors, including ppGpp.

Kinetics of mRNA polyadenylation and its relation to transcription termination in eukaryotes

Processing of most eukaryotic mRNAs includes and polyadenylation of the nascent transcript. Until now there has been no method for the reliable measurement of these processes in vivo. Therefore, in the present work a new technique was developed for measuring precisely the rate of cleavage and polyadenylation in vivo. The method uses a cis-antisense element targeted to an upstream poly (A) signal. Duplex formation of the antisense element with the poly (A) signal region prevents polyadenylation. In a series of expression vector constructs the antisense element was moved increasing distances downstream of its target poly (A) site, reasoning that if it takes the polymerase longer to reach the antisense element, polyadenylation would have more time to occur. Using this method the half time for commitment to cleavage and polyadenylation at the SV40 early poly (A) site and SPA (synthetic poly A site) was found to be 5 seconds. It was found that strong sites (SV 40 late poly (A) site) were processed faster. Commitment to cleavage and polyadenylation was found to be a multistep process. The expression results were confirmed with the help of RNase protection assay. Relationship between polyadenylation and transcription termination was also studied by using G-free assay and found to be positively correlated. Present data support looping moded suggesting some communication exists between polyadenylation complex and RNA pol. II for transcription termination.

Promoter Substitution and Deletion Analysis of Upstream Region Required for rpoS Translational Regulation

ABSTRACT The RpoS sigma factor of enteric bacteria is required for the increased expression of a number of genes that are induced during nutrient limitation and growth into stationary phase and in response to high osmolarity. RpoS is also a virulence factor for several pathogenic species, including Salmonella typhimurium. The activity of RpoS is regulated at both the level of synthesis and protein turnover. Here we investigate the posttranscriptional control of RpoS synthesis by using rpoS-lac protein and operon fusions. Substitution of the native rpoS promoters with the tac orlac UV5 promoters allowed essentially normal regulation after growth into stationary phase in rich medium or after osmotic challenge. Regulation of these fusions required the function ofhfq, encoding the RNA-binding protein host factor I (HF-I). Short deletions from the 5′ end of the rpoS transcript did not affect regulation very much; however, a larger deletion mutation that still retains 220 bp upstream of the rpoS ATG codon, including a proposed antisense element inhibitory for rpoStranslation, was no longer regulated by HF-I. Several models for regulation of rpoS expression by HF-I are discussed.