An RNA species involved in Escherichia coli ribonuclease P activity. Gene cloning and effect on transfer RnA synthesis in vivo

The isolation and characterization of a plasmid capable of complementing the temperature-sensitive transfer RNA biosynthetic mutation rnpA49 (ribonuclease P) is described. The DNA segment responsible for complementation codes for an RNA species, approximately 340 bases long. Hybridization-selection experiments indicated that all rnp mutants were deficient in the production of the complementing RNA at high temperature; these defects were not due to the accumulation of a precursor form of this RNA. Examination of tRNA species synthesized in vivo indicated that the plasmid clone did not completely relieve the deficiency in RNase P activity of rnpA49 since some tRNA precursors still accumulated in strain A49 containing the plasmid. At least one other tRNA precursor was no longer detectable in plasmid-containing rnpA49 cells.     

Two ribosome binding sites from the gene 0.3 messenger RNA of bacteriophage T7

Ribosome-protected regions have been isolated and analyzed from the bacteriophage T7 gene 0.3 mRNA labeled in vivo. Two discrete sites which are nearly equally protected by ribosomes are obtained from what was previously assumed to be a monocistronic message. Use of appropriate T7 deletion mutant RNAs has allowed mapping of both ribosome-recognized regions. Site a is positioned very close to the 5′ terminus of the mRNA and is apparently the initiator region for the major gene 0.3 protein, which acts to overcome the host DNA restriction system. Site b is located within several hundred nucleotides of the 3′ end of the RNA and probably initiates synthesis of a small polypeptide of unknown function. Both ribosome binding sites exhibit features common to other initiator regions from Escherichia coli and bacteriophage mRNAs. The proximity of site a to the RNase III cleavage site at the left end of gene 0.3 may explain why processing by RNase III is required for efficient translation of the major gene 0.3 protein.     

The maize mitochondrial plasmid RNA b is associated with protein during synthesis but is not encapsidated

RNA b is the most abundant member of a family of autonomously replicating single- and double-stranded RNA plasmids found in maize mitochondria. The extent to which this molecule is associated with proteins was investigated by rate zonal and CsCl equilibrium density gradient centrifugation of clarified lysates of S cytoplasm maize mitochondria. A soluble complex of RNA b, responsible for synthesis of the more abundant (+) RNA b strand in mitochondrial lysates, was identified. The complex had a buoyant density of 1.49 g/cm³, indicating a substantial non-nucleic acids content. The sedimentation coefficient of the complex, however, was only slightly larger than that of deproteinized RNA b. Synthesis of RNA b as well as the larger RNA plasmid, RNA a, was resistant to heparin, suggesting that, for both RNAs, preformed complexes between an RNA template and an RNA-dependent RNA polymerase capable of elongating in vivo preinitiated RNA plasmid strands, were present in the lysate. Only a small fraction of RNA b molecules were bound in the complex; the bulk of RNA b sedimented at the same rate as the deproteinized RNA. Thus, after replication, maize mitochondrial plasmids are not associated with nucleoprotein capsids although their synthesis takes place through ribonucleoprotein replication complexes.     

Synthesis and repair of RNA-containing supercoiled plasmid ColE1 DNA in Escherichia coli

Supercoiled plasmid molecules sensitive to nicking by RNase or alkali have been shown to accumulate during replication of colicinogenic factor E1 (ColE1) in Escherichia coli in the presence of chloramphenicol. The possibility that this sensitivity is due to the covalent integration of RNA molecules during the synthesis of plasmid DNA is supported by the demonstration that (a) strands of supercoiled ColE1 newly replicated in the presence of chloramphenicol exhibit sensitivity to RNase and alkali treatment, while (b) RNase- and alkali-resistant circular strands of plasmid DNA synthesized either before or after the addition of chloramphenicol remain resistant during subsequent replication of the plasmid in the presence of chloramphenicol. Furthermore, newly made plasmid DNA strands cannot act as templates for further rounds of replication if they possess an RNA segment. The existence of a repair mechanism for the removal of the RNA segment from supercoiled ColE1 DNA molecules was demonstrated by pulse-chase experiments. It was observed that the proportion of RNase-sensitive molecules is considerably higher in pulse-labeled as compared to continuously labeled ColE1 DNA synthesized in the presence of chloramphenicol, and the proportion of pulse-labeled ColE1 DNA that is RNase sensitive is greatly reduced during a chase period. Removal of the RNA segment is also carried out effectively at the restrictive temperature in temperature-sensitive DNA polymerase I mutants. In a survey of other bacterial mutants defective in the repair of damaged DNA, a substantial increase in the rate of accumulation of RNase-and alkali-sensitive supercoiled ColE1 DNA in the presence of chloramphenicol was observed in recBC and uvrA mutants in comparison with the wild-type strains.     

Air proteins control differential TRAMP substrate specificity for nuclear RNA surveillance

RNA surveillance systems function at critical steps during the formation and function of RNA molecules in all organisms. The RNA exosome plays a central role in RNA surveillance by processing and degrading RNA molecules in the nucleus and cytoplasm of eukaryotic cells. The exosome functions as a complex of proteins composed of a nine-member core and two ribonucleases. The identity of the molecular determinants of exosome RNA substrate specificity remains an important unsolved aspect of RNA surveillance. In the nucleus of Saccharomyces cerevisiae, TRAMP complexes recognize and polyadenylate RNAs, which enhances RNA degradation by the exosome and may contribute to its specificity. TRAMPs contain either of two putative RNA-binding factors called Air proteins. Previous studies suggested that these proteins function interchangeably in targeting the poly(A)-polymerase activity of TRAMPs to RNAs. Experiments reported here show that the Air proteins govern separable functions. Phenotypic analysis and RNA deep-sequencing results from air mutants reveal specific requirements for each Air protein in the regulation of the levels of noncoding and coding RNAs. Loss of these regulatory functions results in specific metabolic and plasmid inheritance defects. These findings reveal differential functions for Air proteins in RNA metabolism and indicate that they control the substrate specificity of the RNA exosome.     

Minor changes largely restore catalytic activity of archaeal RNase P RNA from Methanothermobacter thermoautotrophicus

The increased protein proportion of archaeal and eukaryal ribonuclease (RNase) P holoenzymes parallels a vast decrease in the catalytic activity of their RNA subunits (P RNAs) alone. We show that a few mutations toward the bacterial P RNA consensus substantially activate the catalytic (C-) domain of archaeal P RNA from Methanothermobacter, in the absence and presence of the bacterial RNase P protein. Large increases in ribozyme activity required the cooperative effect of at least two structural alterations. The P1 helix of P RNA from Methanothermobacter was found to be extended, which increases ribozyme activity (ca 200-fold) and stabilizes the tertiary structure. Activity increases of mutated archaeal C-domain variants were more pronounced in the context of chimeric P RNAs carrying the bacterial specificity (S-) domain of Escherichia coli instead of the archaeal S-domain. This could be explained by the loss of the archaeal S-domain''s capacity to support tight and productive substrate binding in the absence of protein cofactors. Our results demonstrate that the catalytic capacity of archaeal P RNAs is close to that of their bacterial counterparts, but is masked by minor changes in the C-domain and, particularly, by poor function of the archaeal S-domain in the absence of archaeal protein cofactors.     

Influence of RNA structural stability on the RNA chaperone activity of the Escherichia coli protein StpA

Proteins with RNA chaperone activity are able to promote folding of RNA molecules by loosening their structure. This RNA unfolding activity is beneficial when resolving misfolded RNA conformations, but could be detrimental to RNAs with low thermodynamic stability. In order to test this idea, we constructed various RNAs with different structural stabilities derived from the thymidylate synthase (td) group I intron and measured the effect of StpA, an Escherichia coli protein with RNA chaperone activity, on their splicing activity in vivo and in vitro. While StpA promotes splicing of the wild-type td intron and of mutants with wild-type-like stability, splicing of mutants with a lower structural stability is reduced in the presence of StpA. In contrast, splicing of an intron mutant, which is not destabilized but which displays a reduced population of correctly folded RNAs, is promoted by StpA. The sensitivity of an RNA towards StpA correlates with its structural stability. By lowering the temperature to 25°C, a temperature at which the structure of these mutants becomes more stable, StpA is again able to stimulate splicing. These observations clearly suggest that the structural stability of an RNA determines whether the RNA chaperone activity of StpA is beneficial to folding.     

An Arabidopsis RNase III-like protein,AtRTL2, cleaves double-stranded RNA in vitro

Class 1 ribonuclease III (RNase III), found in bacteria and yeast, is involved in processing functional RNA molecules such as ribosomal RNAs (rRNAs). However, in Arabidopsis thaliana, the lack of an obvious phenotype or quantitative change in mature rRNAs in class 1 RNase III (AtRTL2) mutants and overexpressing plants suggests that AtRTL2 is not involved in rRNA maturation. We characterized the in vitro activity of AtRTL2 to consider its in vivo function. AtRTL2 cleaved double-stranded RNA (dsRNA) specifically in vitro, yielding products of approximately 25 nt or longer in length, in contrast to 10–20 nt long products in bacteria and yeasts. Although dsRNA-binding activity was not detected, the dsRNA-binding domains in AtRTL2 were essential for its dsRNA-cleaving activity. Accumulation of small RNAs derived from transgene dsRNAs was increased when AtRTL2 was transiently expressed in Nicotiana benthamiana leaves by agroinfiltration. These results raise the possibility that AtRTL2 has functions distinct from those of other class 1 RNase IIIs in vivo.     

Small RNAs of Rous Sarcoma Virus: Characterization by Two-Dimensional Polyacrylamide Gel Electrophoresis and Fingerprint Analysis

Approximately 15 to 20 different species of small (4 to 7S) RNAs have been purified by two-dimensional polyacrylamide gel electrophoresis of RNA isolated from virions of Schmidt-Ruppin D strain of Rous sarcoma virus. Each species of small RNA has been isolated free of 70S RNA; nine of them, including 5S and 7S RNAs, were also found associated with the 70S genomic RNA. Most of the 4S RNAs are present at an average of less than one copy per virion. The 4S RNAs have T1 RNase (EC 2.7.7.26) fingerprints, which are very similar to those of tRNAs. One of the smallest 4S RNAs, which can act as a primer for initiation of RNA-directed DNA synthesis, is associated with the 70S RNA in 1 to 2 copies per complex, whereas an additional 6 to 8 copies of this molecule are free.     

Characterization of RNase R-digested cellular RNA source that consists of lariat and circular RNAs from pre-mRNA splicing

Besides linear RNAs, pre-mRNA splicing generates three forms of RNAs: lariat introns, Y-structure introns from trans-splicing, and circular exons through exon skipping. To study the persistence of excised introns in total cellular RNA, we used three Escherichia coli 3′ to 5′ exoribonucleases. Ribonuclease R (RNase R) thoroughly degrades the abundant linear RNAs and the Y-structure RNA, while preserving the loop portion of a lariat RNA. Ribonuclease II (RNase II) and polynucleotide phosphorylase (PNPase) also preserve the lariat loop, but are less efficient in degrading linear RNAs. RNase R digestion of the total RNA from human skeletal muscle generates an RNA pool consisting of lariat and circular RNAs. RT–PCR across the branch sites confirmed lariat RNAs and circular RNAs in the pool generated by constitutive and alternative splicing of the dystrophin pre-mRNA. Our results indicate that RNase R treatment can be used to construct an intronic cDNA library, in which majority of the intron lariats are represented. The highly specific activity of RNase R implies its ability to screen for rare intragenic trans-splicing in any target gene with a large background of cis-splicing. Further analysis of the intronic RNA pool from a specific tissue or cell will provide insights into the global profile of alternative splicing.     

Metabolism of Ribosomal RNA in Mutants of ESCHERICHIA COLI Doubly Defective in Ribonuclease III and the Transcription Termination Factor Rho

To determine if proteins RNase III and rho, both of which can determine the 3' ends of RNA molecules, can complement each other, double mutants defective in these two factors were constructed. In all cases (four rho mutations tested) the double mutants were viable at lower temperatures, but were unable to grow at higher temperatures at which both of the parental strains grew. Genetic analyses suggested that the combinations of the rnc rho (RNase III-Rho-) mutations was necessary and probably sufficient to confer temperature sensitivity on carrier strains. Physiological studies showed that synthesis and maturation of rRNA, which is greatly affected by RNase III, as well as other RNAs, was indistinguishable in rnc rho strains as compared to rnc rho+ strains, thus suggesting that RNase III and rho do not complement one another in determining the 3' ends of RNA molecules. In rnc rho strains, however, the newly synthesized rRNA failed to accumulate. Thus, decay of rRNA could be the reason for the temperature sensitivity of the double mutant strains. These experiments suggest that RNase III and rho can both protect rRNA from degradation by cellular ribonucleases. They also point to the possibility that the nucleotide sequences involved in the determination of the 3' ends of RNA molecules by these two factors are not identical.     

Decay of RNA in RNA processing mutants of Escherichia coli

Summary Stability of RNA was tested in strains of Escherichia, coli carrying single, double, or triple mutations in the RNA processing enzymes RNase III, RNase E and RNase P. Tests were carried out for total pulse labeled RNA, -galactosidase mRNA and for the decay of preexisting RNA during carbon starvation. Decay of RNA was measured at premissive and nonpermissive temperatures and in no case were significant differences between mutants and non-mutant strains found. Therefore, we conclude that the three processing enzymes; RNase III, E and P do not contribute significantly to turnover of RNA in Escherichia coli.     

A gene affecting accumulation of the RNA moiety of the processing enzyme RNase P.

The level of 10Sb (M1) RNA, the RNA of RNase P, is very low in growing cultures of rnpB mutants. Northern transfer experiments suggested that these strains accumulate no more than 10% of the wild-type level of 10Sb RNA. However, there is no indication that there is a limiting amount of RNase P activity in these mutants in vivo. A plasmid that directs the synthesis of 10Sb RNA does not complement the rnpB mutants, even though there is only a single gene for 10Sb RNA in the Escherichia coli genome. The 10Sb RNA synthesized from this plasmid is equivalent to wild-type 10Sb RNA since it can replace it in the reconstitution of RNase P. The 10Sb RNA, which is a rather stable molecule, is unstable in the presence of the rnpB mutation. This could explain why rnpB mutants do not accumulate 10Sb RNA. An F' plasmid that contains DNA from the rnpB region of the chromosome complements an rnpB mutant in vivo and in vitro, and it also contains the 10Sb RNA gene. A number of possible explanations for these phenomena are discussed.     

CRISPR/Cas9 and TALENs generate heritable mutations for genes involved in small RNA processing of Glycine max and Medicago truncatula

Processing of double‐stranded RNA precursors into small RNAs is an essential regulator of gene expression in plant development and stress response. Small RNA processing requires the combined activity of a functionally diverse group of molecular components. However, in most of the plant species, there are insufficient mutant resources to functionally characterize each encoding gene. Here, mutations in loci encoding protein machinery involved in small RNA processing in soya bean and Medicago truncatula were generated using the CRISPR/Cas9 and TAL‐effector nuclease (TALEN) mutagenesis platforms. An efficient CRISPR/Cas9 reagent was used to create a bi‐allelic double mutant for the two soya bean paralogous Double‐stranded RNA‐binding2 (GmDrb2a and GmDrb2b) genes. These mutations, along with a CRISPR/Cas9‐generated mutation of the M. truncatula Hua enhancer1 (MtHen1) gene, were determined to be germ‐line transmissible. Furthermore, TALENs were used to generate a mutation within the soya bean Dicer‐like2 gene. CRISPR/Cas9 mutagenesis of the soya bean Dicer‐like3 gene and the GmHen1a gene was observed in the T₀ generation, but these mutations failed to transmit to the T₁ generation. The irregular transmission of induced mutations and the corresponding transgenes was investigated by whole‐genome sequencing to reveal a spectrum of non‐germ‐line‐targeted mutations and multiple transgene insertion events. Finally, a suite of combinatorial mutant plants were generated by combining the previously reported Gmdcl1a, Gmdcl1b and Gmdcl4b mutants with the Gmdrb2ab double mutant. Altogether, this study demonstrates the synergistic use of different genome engineering platforms to generate a collection of useful mutant plant lines for future study of small RNA processing in legume crops.     

Functional characterization of the Drosophila MRP (mitochondrial RNA processing) RNA gene

MRP RNA is a noncoding RNA component of RNase mitochondrial RNA processing (MRP), a multi-protein eukaryotic endoribonuclease reported to function in multiple cellular processes, including ribosomal RNA processing, mitochondrial DNA replication, and cell cycle regulation. A recent study predicted a potential Drosophila ortholog of MRP RNA (CR33682) by computer-based genome analysis. We have confirmed the expression of this gene and characterized the phenotype associated with this locus. Flies with mutations that specifically affect MRP RNA show defects in growth and development that begin in the early larval period and end in larval death during the second instar stage. We present several lines of evidence demonstrating a role for Drosophila MRP RNA in rRNA processing. The nuclear fraction of Drosophila MRP RNA localizes to the nucleolus. Further, a mutant strain shows defects in rRNA processing that include a defect in 5.8S rRNA processing, typical of MRP RNA mutants in other species, as well as defects in early stages of rRNA processing.