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At least 325 records · Page 18Linked to original sources
PubMed2006

A germline-specific class of small RNAs binds mammalian Piwi proteins.

Small RNAs associate with Argonaute proteins and serve as sequence-specific guides to regulate messenger RNA stability, protein synthesis, chromatin organization and genome structure. In animals, Argonaute proteins segregate into two subfamilies. The Argonaute subfamily acts in RNA interference and in microRNA-mediated gene regulation using 21-22-nucleotide RNAs as guides. The Piwi subfamily is involved in germline-specific events such as germline stem cell maintenance and meiosis. However, neither the biochemical function of Piwi proteins nor the nature of their small RNA guides is known. Here we show that MIWI, a murine Piwi protein, binds a previously uncharacterized class of approximately 29-30-nucleotide RNAs that are highly abundant in testes. We have therefore named these Piwi-interacting RNAs (piRNAs). piRNAs show distinctive localization patterns in the genome, being predominantly grouped into 20-90-kilobase clusters, wherein long stretches of small RNAs are derived from only one strand. Similar piRNAs are also found in human and rat, with major clusters occurring in syntenic locations. Although their function must still be resolved, the abundance of piRNAs in germline cells and the male sterility of Miwi mutants suggest a role in gametogenesis.

Animals↗
PubMed2001

A mRNA determinant of gRNA-directed kinetoplastid editing.

Several mitochondrial mRNAs of the kinetoplastid protozoa do not encode a functional open reading frame until they have been edited through the addition or deletion of U nucleotides at specific sites. Genetic information specifying the location and extent of editing is present on guide RNAs (gRNAs). The sequence adjacent to most mRNA editing sites has a high purine content which previously has been proposed to facilitate the editing reaction through base-pairing to a poly(U) tail at the 3' end of the gRNA. We demonstrate here that gRNA binding alone is insufficient to create an editing site and that the mRNA sequence near an editing site is an additional determinant affecting the efficiency of the reaction.

Animals↗
PubMed1992

Maxicircle CR1 transcripts of Trypanosoma brucei are edited and developmentally regulated and encode a putative iron-sulfur protein homologous to an NADH dehydrogenase subunit.

The maxicircle of Trypanosoma brucei encodes components of the mitochondrial oxidative phosphorylation system, as do other mitochondrial DNAs, but maxicircle gene identification is complicated by extensive editing of some transcripts. We found that transcripts from the CR1 region were extensively edited, as are other transcripts from maxicircle regions which exhibit strong G versus C strand bias. Editing added 259 uridines and removed 46 uridines to produce an approximately 574-nucleotide mature mRNA. Partially edited cDNAs and potential guide RNAs were also characterized. Initiation and termination codons were created, and they defined an open reading frame encoding a predicted protein of 145 amino acids. This protein contains two iron-sulfur cysteine motifs and is homologous to a subunit of NADH dehydrogenase and to other electron-carrier proteins. Higher levels of both edited and unedited CR1 transcripts accumulated in bloodstream forms of the parasite than in procyclic forms, suggesting developmental regulation of CR1 gene expression.

Amino Acid Sequence↗
PubMed1994

Multiple guide RNAs for identical editing of Trypanosoma brucei apocytochrome b mRNA have an unusual minicircle location and are developmentally regulated.

We identified four different guide RNAs (gRNAs) that specify identical editing of Trypanosoma brucei apocytochrome b (CYb) mRNA, which indicates gRNA redundancy in T. brucei. All four gRNAs appear functional since they occur in chimeras, some of which contain an interesting gRNA 3' "extension." The gRNAs are encoded in different minicircles, rather than maxicircles as in other species. However, these gRNA genes are not between 18-base pair repeats as are the other minicircle gRNA genes in T. brucei. The three minicircles cloned contain the same gRNA genes, one of which is substantially diverged, all in the same order, indicating that they are related. CYb gRNA is less abundant in procyclic than bloodstream forms. Procyclic forms contain abundant edited CYb mRNA unlike bloodstream forms thus suggesting that CYb mRNA editing may be regulated at the level of gRNA utilization.

Animals↗
PubMed2001

Guide RNAs of the recently isolated LEM125 strain of Leishmania tarentolae: an unexpected complexity.

Guide RNAs (gRNAs) are encoded both in the maxicircle and minicircle components of the mitochondrial DNA of trypanosomatid protozoa. These RNAs mediate the precise insertion and deletion of U residues in transcripts of the maxicircle DNA. We showed previously that the old UC laboratory strain of Leishmania tarentolae apparently lost more than 40 minicircle-encoded gRNAs that are present in the recently isolated LEM125 strain (Thiemann et al., EMBO J, 1994, 13:5689-5700]. We have further analyzed the population of minicircle-encoded gRNAs in the LEM125 strain. Sau3AI and MspI minicircle libraries were constructed and screened for novel gRNAs by negative colony hybridization. This search yielded 20 minicircles encoding new gRNAs that covered most of the remaining gaps in the editing cascades of the ND8, ND9, G4, and G5 genes, and in addition, more than 30 minicircles containing either unassigned or undetectable gRNA genes. We also completely sequenced 34 of the 45 minicircle sequence classes encoding previously identified gRNAs. A total of 19 pairs of redundant gRNAs, which are gRNAs of different sequences covering the same editing blocks, were identified. The gRNAs in each redundant pair generally had different relative abundances and different extents of mismatches with edited sequences. Alignments of the minicircles encoding redundant gRNAs yielded 59 to 93% matching nucleotides, suggesting an origin from duplication of ancestral minicircles and subsequent genetic drift. We propose a functional explanation for the existence of redundant gRNAs in this strain.

Amino Acid Sequence↗
PubMed2004

Uncoupling of RNAi from active translation in mammalian cells.

Small inhibitory RNAs (siRNAs) are produced from longer RNA duplexes by the RNAse III family member Dicer. The siRNAs function as sequence-specific guides for RNA cleavage or translational inhibition. The precise mechanism by which siRNAs direct the RNA-induced silencing complex (RISC) to find the complementary target mRNA remains a mystery. Some biochemical evidence connects RNAi with translation making attractive the hypothesis that RISC is coupled with the translational apparatus for scanning mRNAs. Such coupling would facilitate rapid alignment of the siRNA antisense with the complementary target sequence. To test this hypothesis we took advantage of a well-characterized translational switch afforded by the ferritin IRE-IRP to analyze RNAi mediated cleavage of a target mRNA in the presence and absence of translation. Our results demonstrate that neither active translation nor unidirectional scanning is required for siRNA mediated target degradation. Our findings demonstrate that nontranslated mRNAs are highly susceptible to RNAi, and blocking scanning from both the 5' and 3' ends of an mRNA does not impede RNAi. Interestingly, RNAi is about threefold more active in the absence of translation.

Base Sequence↗
PubMed2004

Structural basis for overhang-specific small interfering RNA recognition by the PAZ domain.

Short RNAs mediate gene silencing, a process associated with virus resistance, developmental control and heterochromatin formation in eukaryotes. RNA silencing is initiated through Dicer-mediated processing of double-stranded RNA into small interfering RNA (siRNA). The siRNA guide strand associates with the Argonaute protein in silencing effector complexes, recognizes complementary sequences and targets them for silencing. The PAZ domain is an RNA-binding module found in Argonaute and some Dicer proteins and its structure has been determined in the free state. Here, we report the 2.6 A crystal structure of the PAZ domain from human Argonaute eIF2c1 bound to both ends of a 9-mer siRNA-like duplex. In a sequence-independent manner, PAZ anchors the 2-nucleotide 3' overhang of the siRNA-like duplex within a highly conserved binding pocket, and secures the duplex by binding the 7-nucleotide phosphodiester backbone of the overhang-containing strand and capping the 5'-terminal residue of the complementary strand. On the basis of the structure and on binding assays, we propose that PAZ might serve as an siRNA-end-binding module for siRNA transfer in the RNA silencing pathway, and as an anchoring site for the 3' end of guide RNA within silencing effector complexes.

Amino Acid Motifs↗
PubMed2000

In vivo inhibition by a site-specific catalytic RNA subunit of RNase P designed against the BCR-ABL oncogenic products: a novel approach for cancer treatment.

One major obstacle to the effective treatment of cancer is to distinguish between tumor cells and normal cells. The chimeric molecules created by cancer-associated chromosomal abnormalities are ideal therapeutic targets because they are unique to the disease. We describe the use of a novel approach based on the catalytic RNA subunit of RNase P to destroy specifically the tumor-specific fusion genes created as a result of chromosome abnormalities. Using as a target model the abnormal BCR-ABL p190 and p210 products, we constructed M1-RNA with guide sequences that recognized the oncogenic messengers at the fusion point (M1-p190-GS and M1-p210-GS). To test the effectiveness and the specificity of M1-p190-GS and M1-p210-GS, we studied in vitro and in vivo effects of these RNA enzymes against BCR-ABL(p190) and BCR-ABL(p210), bearing in mind that both fusion genes share the ABL sequence but differ in the sequence coming from the BCR gene. We showed that M1-p190-GS and M1-p210-GS can act as sequence-specific endonucleases and can exclusively cleave target RNA that forms a base pair with the guide sequence (GS). We also demonstrated that when M1-p190-GS and M1-p210-GS were expressed in proper mammalian cell models, they abolished the effect of BCR-ABL by specifically decreasing the amount of the target BCR-ABL mRNA and preventing the function of the BCR-ABL oncogenes. These data clearly demonstrate the usefulness of the catalytic activity of M1-GS RNA to cleave specifically the chimeric molecules created by chromosomal abnormalities in human cancer and to represent a novel approach to cancer treatment.

Animals↗
PubMed1976

The effect of ribonuclease on the penetration of R17 phage A-protein and RNA.

In an attempt to throw further light on the relationship of R17 phage RNA and A-protein during the early stages of infection, studies were carried out to determine the effect of ribonuclease (ribonuclease I, EC 3.1.4.22) on the ability of these two phage components to penetrate into host bacteria. It was found that the penetration of phage RNA is affected by ribonuclease concentrations as low as 0.1 mug/ml, while the penetration of phage A-protein was unaffected by ribonuclease concentrations as high as 20 mug/ml. In addition, it was found that a significant fraction of the phage RNA is resistant to the ribonuclease effect. This RNase-resistant portion of the phage population increased with increasing phage concentrations, and gave rise to the penetration of intact, 28S RNA molecules that produced the expected number of infectious centers. These findings are discussed in terms of a model for phage RNA injection in which the A-protein functions both as an attachment organelle and a pilot protein that guides the RNA from the capsid to the exterior surface of the F pilus, and thence into the host bacterium.

Coliphages↗
PubMed1999

In vitro uridine insertion RNA editing mediated by cis-acting guide RNAs.

Uridine (U) insertion/deletion editing of mitochondrial mRNAs in kinetoplastid protozoa is a posttranscriptional process mediated by guide RNAs (gRNAs). The gRNAs direct the precise insertion and deletion of Us by a cleavage-ligation mechanism involving base pairing. We show that a cognate gRNA in cis at the 3' end of a preedited NADH dehydrogenase 7 (ND7) mRNA substrate can direct U insertions at editing site 1 when incubated with a mitochondrial lysate from Leishmania tarentolae. The efficiency of gRNA-dependent U insertion mediated by a cis-acting gRNA is greater on a molar basis than that for a trans-acting gRNA, as expected for a unimolecular gRNA:mRNA interaction. Blocking the 3' end of a cis-acting gRNA lacking a 3' oligo[U] tail has no effect on gRNA-dependent U insertions, nor does providing the gRNA in cis upstream of the mRNA, confirming the previous observation that the terminal 2'- and 3'-hydroxyls of the gRNA are not involved in U insertion activity. These results also establish that the oligo[U] tail is not required for U insertion in vitro. Increasing the extent of base pairing between the 3' end of the gRNA and the 5' end of the mRNA significantly increases in vitro gRNA-dependent U insertion at site 1, presumably by maintaining the mRNA 5' cleavage fragment within the editing complex. We speculate that, in vivo, protein:RNA and/or protein:protein interactions may be responsible for maintaining the mRNA 5' cleavage fragment in close proximity to the mRNA 3' cleavage fragment, and that such interactions may be rate limiting in vitro.

Animals↗
PubMed2006

The role of positively charged amino acids and electrostatic interactions in the complex of U1A protein and U1 hairpin II RNA.

Previous kinetic investigations of the N-terminal RNA recognition motif (RRM) domain of spliceosomal protein U1A, interacting with its RNA target U1 hairpin II, provided experimental evidence for a 'lure and lock' model of binding in which electrostatic interactions first guide the RNA to the protein, and close range interactions then lock the two molecules together. To further investigate the 'lure' step, here we examined the electrostatic roles of two sets of positively charged amino acids in U1A that do not make hydrogen bonds to the RNA: Lys20, Lys22 and Lys23 close to the RNA-binding site, and Arg7, Lys60 and Arg70, located on 'top' of the RRM domain, away from the RNA. Surface plasmon resonance-based kinetic studies, supplemented with salt dependence experiments and molecular dynamics simulation, indicate that Lys20 predominantly plays a role in association, while nearby residues Lys22 and Lys23 appear to be at least as important for complex stability. In contrast, kinetic analyses of residues away from the RNA indicate that they have a minimal effect on association and stability. Thus, well-positioned positively charged residues can be important for both initial complex formation and complex maintenance, illustrating the multiple roles of electrostatic interactions in protein-RNA complexes.

Amino Acid Sequence↗
PubMed2001

RNA interference in Trypanosoma brucei: cloning of small interfering RNAs provides evidence for retroposon-derived 24-26-nucleotide RNAs.

In animals and protozoa, gene-specific double-stranded RNA (dsRNA) triggers degradation of homologous cellular RNAs, a phenomenon known as RNA interference (RNAi). In vitro and in vivo dsRNA is processed by a nuclease to produce 21-25-nt small interfering RNAs (siRNAs) that guide target RNA degradation. Here we show that activation of RNAi in Trypanosoma bruceiby expression or electroporation of actin dsRNA results in production of actin siRNAs and that 10% of these RNAs sediment as high-molecular-weight complexes at 100,000 x g. To characterize actin siRNAs, we established a cloning and enrichment strategy starting from 20-30 nt RNAs isolated from high-speed pellet and supernatant fractions. Sequence analysis revealed that actin siRNAs are 24-26 nt long and their distribution relative to actin dsRNA was similar in the two fractions. By sequencing over 1,300 fragments derived from the high-speed pellet fraction RNA, we found abundant 24-26-nt-long fragments homologous to the ubiquitous retroposon INGI and the site-specific retroposon SLACS. Northern hybridization with strand-specific probes confirmed that retroposon-derived 24-26-nt RNAs are present in both supernatant and high-speed pellet fractions and that they are constitutively expressed. We speculate that RNAi in trypanosomes serves a housekeeping function and is likely to be involved in silencing retroposon transcripts.

Actins↗
PubMed1992

RNA editing in trypanosomes. The us(e) of guide RNAs.

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.

Animals↗
PubMed2004

RNA interference: the molecular immune system.

Introduction of double-stranded RNA (dsRNA) into cells expressing a homologous gene triggers RNA interference (RNAi), or RNA-based gene silencing (RBGS). The dsRNA degrades corresponding host mRNA into small interfering RNAs (siRNAs) by a protein complex containing Dicer. siRNAs in turn are incorporated into the RNA-induced silencing complex (RISC) that includes helicase, RecA, and exo- and endo-nucleases as well as other proteins. Following its assembly, the RISC guides the RNA degradation machinery to the target RNAs and cleaves the cognate target RNA in a sequence-specific, siRNA-dependent manner. RNAi has now been documented in a wide variety of organisms, including plants, fungi, flies, worms, and more recently, higher mammals. In eukaryotes, dsRNA directed against a range of viruses (i.e., HIV-1, RSV, HPV, poliovirus and others) and endogenous genes can induce sequence-specific inhibition of gene expression. In invertebrates, RNAi can be efficiently triggered by either long dsRNAs or 21- to 23-nt-long siRNAs. However, in jawed vertebrates, dsRNA longer than 30 bp can induce interferon and thus trigger undesirable side effects instead of initiating RNAi. siRNAs have been shown to act as potent inducers of RNAi in cultured mammalian cells. Many investigators have suggested that siRNAs may have evolved as a normal defense against endogenous and exogenous transposons and retroelements. Through a combination of genetic and biochemical approaches, some of the mechanisms underlying RNAi have been described. Recent data in C. elegans shows that two homologs of siRNAs, microRNAs (miRNAs) and tiny noncoding RNAs (tncRNAs) are endogenously expressed. However, many aspects of RNAi-induced gene silencing, including its origins and the selective pressures which maintain it, remain undefined. Its evolutionary history may pass through the more primitive immune functions of prokaryotes involving restriction enzymes that degrade plasmid DNA molecules that enter bacterial cells. RNAi has evolved further among eukaryotes, in which its wide distribution suggests early origins. RNAi seems to be involved in a variety of regulatory and immune functions that may differ among various kingdoms and phyla. We present here proposed mechanisms by which RBGS protects the host against endogenous and exogenous transposons and retroelements. The potential for therapeutic application of RBGS technology in treating viral infections such as HIV is also discussed.

AIDS Vaccines↗
PubMed2001

The complete DNA sequence of the mitochondrial genome of Physarum polycephalum.

The complete sequence of the mitochondrial DNA (mtDNA) of the true slime mold Physarun polycephalum has been determined. The mtDNA is a circular 62,862-bp molecule with an A+T content of 74.1%. A search with the program BLAST X identified the protein-coding regions. The mitochondrial genome of P. polycephalum was predicted to contain genes coding for 12 known proteins [for three cytochrome c oxidase subunits, apocytochrome b, two F1Fo-ATPase subunits, five NADH dehydrogenase (nad) subunits, and one ribosomal protein], two rRNA genes, and five tRNA genes. However, the predicted ORFs are not all in the same frame, because mitochondrial RNA in P. polycephalum undergoes RNA editing to produce functional RNAs. The nucleotide sequence of an nad7 cDNA showed that 51 nucleotides were inserted at 46 sites in the mRNA. No guide RNA-like sequences were observed in the mtDNA of P. polycephalum. Comparison with reported Physarum mtDNA sequences suggested that sites of RNA editing vary among strains. In the Physarum mtDNA, 20 ORFs of over 300 nucleotides were found and ORFs 14 19 are transcribed.

Amino Acid Sequence↗
PubMed1992

Guide RNAs for transcripts with developmentally regulated RNA editing are present in both life cycle stages of Trypanosoma brucei.

RNA editing of several mitochondrial transcripts in Trypanosoma brucei is developmentally regulated. The cytochrome b and cytochrome oxidase II mRNAs are edited in procyclic-form parasites but are primarily unedited in bloodstream forms. The latter forms lack the mitochondrial respiratory system present in procyclic forms. Editing of the NADH dehydrogenase 7 (ND7) and ND8 transcripts is also developmentally regulated but occurs preferentially in bloodstream forms. Other transcripts, cytochrome oxidase III and ATPase 6, are edited in both life forms. We have identified many minicircle-encoded guide RNAs (gRNAs) for ATPase 6, ND7, and ND8. The characteristics of these gRNAs reveal how extensively edited RNA can be edited in the 3'-to-5' direction. Northern (RNA) blot and primer extension analyses indicate that gRNAs for transcripts whose editing is developmentally regulated are present in both procyclic and bloodstream form parasites. These results suggest that the developmental regulation of editing in these transcripts is not controlled by the presence or absence of gRNAs.

Aging↗
PubMed2006

Structure-guided mutational analysis of T4 RNA ligase 1.

T4 RNA ligase 1 (Rnl1) is a tRNA repair enzyme that circumvents an RNA-damaging host antiviral response. Whereas the three-step reaction scheme of Rnl1 is well established, the structural basis for catalysis has only recently been appreciated as mutational and crystallographic approaches have converged. Here we performed a structure-guided alanine scan of nine conserved residues, including side chains that either contact the ATP substrate via adenine (Leu179, Val230), the 2'-OH (Glu159), or the gamma phosphate (Tyr37) or coordinate divalent metal ions at the ATP alpha phosphate (Glu159, Tyr246) or beta phosphate (Asp272, Asp273). We thereby identified Glu159 and Tyr246 as essential for RNA sealing activity in vitro and for tRNA repair in vivo. Structure-activity relationships at Glu159 and Tyr246 were clarified by conservative substitutions. Eliminating the phosphate-binding Tyr37, and the magnesium-binding Asp272 and Asp273 side chains had little impact on sealing activity in vitro or in vivo, signifying that not all atomic interactions in the active site are critical for function. Analysis of mutational effects on individual steps of the ligation pathway underscored how different functional groups come into play during the ligase-adenylylation reaction versus the subsequent steps of RNA-adenylylation and phosphodiester formation. Moreover, the requirements for sealing exogenous preformed RNA-adenylate are more stringent than are those for sealing the RNA-adenylate intermediate formed in situ during ligation of a 5'-PO4 RNA.

Adenosine Monophosphate↗
PubMed2025

Determining the biochemical function of type IV CRISPR ribonucleoprotein complexes and accessory proteins.

Type IV CRISPR systems are phylogenetically diverse and poorly understood. However, recently, major strides have been made toward understanding type IV-A systems. In type IV-A systems, a multi-subunit ribonucleoprotein complex, called the Csf complex, uses a CRISPR-derived guide to bind double-stranded DNA, forming an R-loop to which a helicase called CRISPR-associated DinG (CasDinG) is recruited. It is proposed that the ATP-dependent helicase activity of CasDinG then unwinds duplex DNA near the targeting site, impairing RNA transcription, and gene expression. Here we describe methods used to investigate the type IV-A system from Pseudomonas aeruginosa strain 83 including a plasmid clearance assay, expression and purification of type IV ribonucleoprotein complexes and proteins, nucleic acid binding assays, and CasDinG helicase assays. These methods provide a foundation for future work aimed at understanding these enigmatic systems.

Ribonucleoproteins↗