C. elegans and H. sapiens mRNAs with edited 3' UTRs are present on polysomes

Adenosine deaminases that act on RNA (ADARs) are editing enzymes that convert adenosine to inosine in double-stranded RNA (dsRNA). ADARs sometimes target codons so that a single mRNA yields multiple protein isoforms. However, ADARs most often target noncoding regions of mRNAs, such as untranslated regions (UTRs). To understand the function of extensive double-stranded 3′ UTR structures, and the inosines within them, we monitored the fate of reporter and endogenous mRNAs that include structured 3′ UTRs in wild-type Caenorhabditis elegans and in strains with mutations in the ADAR genes. In general, we saw little effect of editing on stability or translatability of mRNA, although in one case an ADR-1 dependent effect was observed. Importantly, whereas previous studies indicate that inosine-containing RNAs are retained in the nucleus, we show that both C. elegans and Homo sapiens mRNAs with edited, structured 3′ UTRs are present on translating ribosomes.     

Distinct roles for sequences upstream of and downstream from Physarum editing sites

RNAs in the mitochondria of Physarum polycephalum contain nonencoded nucleotides that are added during RNA synthesis. Essentially all steady-state RNAs are accurately and fully edited, yet the signals guiding these precise nucleotide insertions are presently unknown. To localize the regions of the template that are required for editing, we constructed a series of chimeric templates that substitute varying amounts of DNA either upstream of or downstream from C insertion sites. Remarkably, all sequences necessary for C addition are contained within ∼9 base pairs on either side of the insertion site. In addition, our data strongly suggest that sequences within this critical region affect different steps in the editing reaction. Template alterations upstream of an editing site influence nucleotide selection and/or insertion, while downstream changes affect editing site recognition and templated extension from the added, unpaired nucleotide. The data presented here provide the first evidence that individual regions of the DNA template play discrete mechanistic roles and represent a crucial initial step toward defining the source of the editing specificity in Physarum mitochondria. In addition, these findings have mechanistic implications regarding the potential involvement of the mitochondrial RNA polymerase in the editing reaction.     

Conditional gene silencing of multiple genes with antisense RNAs and generation of a mutator strain of Escherichia coli

In this study, we describe a method of simultaneous conditional gene silencing of up to four genes in Escherichia coli by using antisense RNAs. We used antisense RNAs with paired termini, which carried flanking inverted repeats to create paired double-stranded RNA termini; these RNAs have been proven to have high silencing efficacy. To express antisense RNAs, we constructed four IPTG-inducible vectors carrying different but compatible replication origins. When the lacZ antisense RNA was expressed using these vectors, lacZ expression was successfully silenced by all the vectors, but the expression level of the antisense RNA and silencing efficacy differed depending on the used vectors. All the vectors were co-transformable; the antisense RNAs against lacZ, ackA, pta and pepN were co-expressed, and silencing of all the target genes was confirmed. Furthermore, when antisense RNAs were targeted to the mutator genes mutS, mutD (dnaQ) and ndk, which are involved in DNA replication or DNA mismatch repair, spontaneous mutation frequencies increased over 2000-fold. The resulting mutator strain is useful for random mutagenesis of plasmids. The method provides a robust tool for investigating functional relationships between multiple genes or altering cell phenotypes for biotechnological and industrial applications.     

A simple and universal ligation mediated fusion of genes based on hetero-staggered PCR for generating immunodominant chimeric proteins

A potential relationship between transposon-derived repeats (TDR) and human germline methylation is of biological importance since many genes are flanked by TDR and methylation could affect the expression of nearby genes. Furthermore, DNA methylation has been suggested as a global defense mechanism against genome instability threatened by TDR. We studied the correlation between the density of HapMap methyl-associated SNPs (mSNPs), a marker of germline methylation, and proportion of TDR.After correcting for confounding variables, we found a negative correlation between proportion of Alu repeats and mSNP density for 125-1000 kb windows. Similar results were found for the most active subgroup of repeats. In contrast, a negative correlation between proportion of L1 repeats and mSNP density was found only in the larger 1000 kb windows.Using methylation data on germ cells (sperm) from the Human Epigenome Project, we found a lower proportion of Alu repeats adjacent (3-15 kb) to hypermethylated amplicons. On the contrary, there was a higher proportion of L1 repeats in the 3-5 kb of sequence flanking hypermethylated amplicons but not in the 10-15 kb flanks.Our data indicate a differential response to the major repeat families and that DNA methylation is unlikely to be a uniform global defense system against all TDR. It appears to play a role for the L1 subgroup, with sequences adjacent to L1 repeats methylated in response to their proximity. In contrast, sequences adjacent to Alu repeats appear to be hypomethylated, arguing against a role of methylation in germline defense against those elements.     

Arabidopsis thaliana endonuclease V is a ribonuclease specific for inosine-containing single-stranded RNA

Endonuclease V is highly conserved, both structurally and functionally, from bacteria to humans, and it cleaves the deoxyinosine-containing double-stranded DNA in Escherichia coli, whereas in Homo sapiens it catalyses the inosine-containing single-stranded RNA. Thus, deoxyinosine and inosine are unexpectedly produced by the deamination reactions of adenine in DNA and RNA, respectively. Moreover, adenosine-to-inosine (A-to-I) RNA editing is carried out by adenosine deaminase acting on dsRNA (ADARs). We focused on Arabidopsis thaliana endonuclease V (AtEndoV) activity exhibiting variations in DNA or RNA substrate specificities. Since no ADAR was observed for A-to-I editing in A. thaliana, the possibility of inosine generation by A-to-I editing can be ruled out. Purified AtEndoV protein cleaved the second and third phosphodiester bonds, 3′ to inosine in single-strand RNA, at a low reaction temperature of 20–25°C, whereas the AtEndoV (Y100A) protein bearing a mutation in substrate recognition sites did not cleave these bonds. Furthermore, AtEndoV, similar to human EndoV, prefers RNA substrates over DNA substrates, and it could not cleave the inosine-containing double-stranded RNA. Thus, we propose the possibility that AtEndoV functions as an RNA substrate containing inosine induced by RNA damage, and not by A-to-I RNA editing in vivo.     

Alu RNP and Alu RNA regulate translation initiation in vitro

Alu elements are the most abundant repetitive elements in the human genome; they emerged from the signal recognition particle RNA gene and are composed of two related but distinct monomers (left and right arms). Alu RNAs transcribed from these elements are present at low levels at normal cell growth but various stress conditions increase their abundance. Alu RNAs are known to bind the cognate proteins SRP9/14. We purified synthetic Alu RNP, composed of Alu RNA in complex with SRP9/14, and investigated the effects of Alu RNPs and naked Alu RNA on protein translation. We found that the dimeric Alu RNP and the monomeric left and right Alu RNPs have a general dose-dependent inhibitory effect on protein translation. In the absence of SRP9/14, Alu RNA has a stimulatory effect on all reporter mRNAs. The unstable structure of sRight RNA suggests that the differential activities of Alu RNP and Alu RNA may be explained by conformational changes in the RNA. We demonstrate that Alu RNPs and Alu RNAs do not stably associate with ribosomes during translation and, based on the analysis of polysome profiles and synchronized translation, we show that Alu RNP and Alu RNA regulate translation at the level of initiation.     

RNA editing in trypanosomes

Guide RNAs are encoded in maxicircle and minicircle DNA of trypanosome mitochondria. They play a pivotal role in RNA editing, a process during which the nucleotide sequence of mitochondrial RNAs is altered by U-insertion and deletion. Guide RNAs vary in length from 35 to 78 nucleotides, which correlates with the variation in length of the three functionally important regions of which they are composed: (i) a 4–14 nucleotide anchor sequence embedded in the 5 region, which is complementary to a target sequence on the pre-edited RNA downstream of an editing domain, (ii) a middle part containing the editing information, which ranges from guiding the insertion of just one U into one site to that of the insertion of 32 Us into 10 sites, and (iii) a 5–24 nucleotide 3 terminal oligo [U] extension. Moreover, a variable uridylation site creates gRNAs containing a varying segment of editing information for the same domain. Comparison of different guide RNAs demonstrates that, besides the U-tail, they have no obvious common primary and secondary sequence motifs, each particular sequence being unique. The occurrence in vivo and the synthesis in vitro of chimeric molecules, in which a guide RNA is covalently linked through its 3 U-tail to an editing site of a pre-edited RNA, suggests that RNA editing occurs by consecutive transesterification reactions and is evidence that the guide RNAs not only provide the genetic information, but also the Us themselves.Abbreviations gRNA guide RNA     

Identification of the Active Sites in the Methyltransferases of a Transcribing dsRNA Virus

Many double-stranded RNA (dsRNA) viruses are capable of transcribing and capping RNA within a stable icosahedral viral capsid. The turret of turreted dsRNA viruses belonging to the family Reoviridae is formed by five copies of the turret protein, which contains domains with both 7-N-methyltransferase and 2′-O-methyltransferase activities, and serves to catalyze the methylation reactions during RNA capping. Cypovirus of the family Reoviridae provides a good model system for studying the methylation reactions in dsRNA viruses. Here, we present the structure of a transcribing cypovirus to a resolution of ~ 3.8 Å by cryo-electron microscopy. The binding sites for both S-adenosyl-l-methionine and RNA in the two methyltransferases of the turret were identified. Structural analysis of the turret in complex with RNA revealed a pathway through which the RNA molecule reaches the active sites of the two methyltransferases before it is released into the cytoplasm. The pathway shows that RNA capping reactions occur in the active sites of different turret protein monomers, suggesting that RNA capping requires concerted efforts by at least three turret protein monomers. Thus, the turret structure provides novel insights into the precise mechanisms of RNA methylation.