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At least 217 records · Page 12Linked to original sources
PubMed1998

Purification and characterization of MAR1. A mitochondrial associated ribonuclease from Leishmania tarentolae.

A relatively thermostable 22-kDa endoribonuclease (MAR1) was purified more than 10,000-fold from a mitochondrial extract of Leishmania tarentolae and the gene cloned. The purified nuclease has a Km of 100-145 +/- 33 nM and a Vmax of 1.8-2.9 +/- 2 nmol/min, depending on the RNA substrate, and yields a 3'-OH and a 5'-phosphate. Cleavage was limited to several specific sites in the substrate RNAs tested, but cleavage of pre-edited RNAs was generally independent of the addition of cognate guide RNA. The MAR1 gene was expressed in Escherichia coli or in L. tarentolae cells, and the recombinant protein was affinity-purified. The cleavage specificity of the recombinant enzyme from L. tarentolae was identical to that of the native enzyme. The single copy MAR1 gene maps to an 820-kilobase pair chromosome and contains an open reading frame of 579 nucleotides. The 18-amino acid N-terminal sequence shows characteristics of an uncleaved mitochondrial targeting sequence. Data base searching revealed two homologues of MAR1 corresponding to unidentified open reading frames in Caenorhabditis elegans (GenBankTM accession number Z69637) and Archaeoglobus fulgidus (GenBankTM accession number AE000943). The function of MAR1 in mitochondrial RNA metabolism in L. tarentolae remains to be determined.

Amino Acid Sequence↗
PubMed2006

Analysis of the binding of the N-terminal conserved domain of yeast Cbf5p to a box H/ACA snoRNA.

During ribosome biogenesis, the RNA precursor to mature rRNAs undergoes numerous post-transcriptional chemical modifications of bases, including conversions of uridines to pseudouridines. In archaea and eukaryotes, these conversions are performed by box H/ACA small ribonucleoprotein particles (box H/ACA RNPs), which contain a small guide RNA responsible for the selection of substrate uridines and four proteins, including the pseudouridine synthase, Cbf5p. So far, no in vitro reconstitution of eukaryotic box H/ACA RNPs from purified components has been achieved, principally due to difficulties in purifying recombinant eukaryotic Cbf5p. In this study, we present the purification of a truncated derivative of yeast Cbf5p (Cbf5(Delta)p) that retains the highly conserved TRUB and PUA domains. We have used band retardation assays to show that Cbf5(Delta)p on its own binds to box H/ACA small nucleolar (sno)RNAs. We demonstrate that the conserved H and ACA boxes enhance the affinity of the protein for the snoRNA. Furthermore, like its archaeal homologs, Cbf5(Delta)p can bind to a single stem-loop-box ACA RNA. Finally, we report the first enzymatic footprinting analysis of a Cbf5-RNA complex. Our results are compatible with the view that two molecules of Cbf5p interact with a binding platform constituted by the 5' end of the RNA, the single-stranded hinge domain containing the conserved H box, and the 3' end of the molecule, including the conserved ACA box.

Amino Acid Sequence↗
PubMed2026

Protocol to identify genes required for cardiomyocyte development using Perturb-Seq.

While Perturb-Seq combines CRISPR-based screening with single-cell RNA sequencing (scRNA-seq), large-scale experiments are costly and its application during development is complicated by differentiation heterogeneity. Here, we present a protocol to identify genes required for cardiomyocyte development using Perturb-Seq. We describe steps for sgRNA (single guide RNA) library cloning and infection, cardiomyocyte differentiation, cell hashing, super loading, and scRNA-seq. We then detail procedures for sequencing, mapping, and data analysis. For complete details on the use and execution of this protocol, please refer to Sivakumar et al.1.

CRISPR↗
PubMed2006

On the origin and functions of RNA-mediated silencing: from protists to man.

Double-stranded RNA has been shown to induce gene silencing in diverse eukaryotes and by a variety of pathways. We have examined the taxonomic distribution and the phylogenetic relationship of key components of the RNA interference (RNAi) machinery in members of five eukaryotic supergroups. On the basis of the parsimony principle, our analyses suggest that a relatively complex RNAi machinery was already present in the last common ancestor of eukaryotes and consisted, at a minimum, of one Argonaute-like polypeptide, one Piwi-like protein, one Dicer, and one RNA-dependent RNA polymerase. As proposed before, the ancestral (but non-essential) role of these components may have been in defense responses against genomic parasites such as transposable elements and viruses. From a mechanistic perspective, the RNAi machinery in the eukaryotic ancestor may have been capable of both small-RNA-guided transcript degradation as well as transcriptional repression, most likely through histone modifications. Both roles appear to be widespread among living eukaryotes and this diversification of function could account for the evolutionary conservation of duplicated Argonaute-Piwi proteins. In contrast, additional RNAi-mediated pathways such as RNA-directed DNA methylation, programmed genome rearrangements, meiotic silencing by unpaired DNA, and miRNA-mediated gene regulation may have evolved independently in specific lineages.

Animals↗
PubMed2025

Mutational scanning of TnpB reveals latent activity for genome editing.

TnpB is a diverse family of RNA-guided endonucleases associated with prokaryotic transposons. Due to their small size and putative evolutionary relationship to CRISPR-Cas12, TnpB enzymes hold significant potential for genome editing. However, most TnpBs lack robust gene editing activity, and unbiased profiling of mutational effects on editing activity has not been explored. Here, we mapped comprehensive sequence-function landscapes of a TnpB ribonucleoprotein and discovered many activating mutations in both the protein and RNA. One- and two-position RNA mutants outperform existing variants, highlighting the utility of systematic RNA scaffold mutagenesis. Leveraging the protein's mutational landscape, we identified enhanced TnpB variants from a combinatorial library of activating mutations. These variants enhanced editing in human cells, N. benthamiana, pepper, and rice, with up to a fifty-fold increase compared to wild-type TnpB. These findings highlight previously unknown elements critical for regulating TnpB endonuclease activity and reveal surprising latent activity accessible through mutation.

Journal Article↗
PubMed2001

Non-coding RNA genes and the modern RNA world.

Non-coding RNA (ncRNA) genes produce functional RNA molecules rather than encoding proteins. However, almost all means of gene identification assume that genes encode proteins, so even in the era of complete genome sequences, ncRNA genes have been effectively invisible. Recently, several different systematic screens have identified a surprisingly large number of new ncRNA genes. Non-coding RNAs seem to be particularly abundant in roles that require highly specific nucleic acid recognition without complex catalysis, such as in directing post-transcriptional regulation of gene expression or in guiding RNA modifications.

Base Sequence↗
PubMed2004

Involvement of putative SNF2 chromatin remodeling protein DRD1 in RNA-directed DNA methylation.

In plants, the mechanism by which RNA can induce de novo cytosine methylation of homologous DNA is poorly understood. Cytosines in all sequence contexts become modified in response to RNA signals. Recent work has implicated the de novo DNA methyltransferases (DMTases), DRM1 and DRM2, in establishing RNA-directed methylation of the constitutive nopaline synthase promoter, as well as the DMTase MET1 and the putative histone deacetylase HDA6 in maintaining or enhancing CpG methylation induced by RNA. Despite the identification of enzymes that catalyze epigenetic modifications in response to RNA signals, it is unclear how RNA targets DNA for methylation. A screen for mutants defective in RNA-directed DNA methylation identified a novel putative chromatin-remodeling protein, DRD1. This protein belongs to a previously undefined, plant-specific subfamily of SWI2/SNF2-like proteins most similar to the RAD54/ATRX subfamily. In drd1 mutants, RNA-induced non-CpG methylation is almost eliminated at a target promoter, resulting in reactivation, whereas methylation of centromeric and rDNA repeats is unaffected. Thus, unlike the SNF2-like proteins DDM1/Lsh1 and ATRX, which regulate methylation of repetitive sequences, DRD1 is not a global regulator of cytosine methylation. DRD1 is the first SNF2-like protein implicated in an RNA-guided, epigenetic modification of the genome.

Amino Acid Sequence↗
PubMed2006

Structural and biochemical advances in mammalian RNAi.

RNAi is a collection of processes mediated by small RNAs that silence gene expression in a sequence-specific manner. Studies of processes as divergent as post-transcriptional gene silencing, transcriptional silencing through RNA-directed DNA methylation, or heterochromatin formation, and even RNA-guided DNA elimination have converged on a core pathway. This review will highlight recent structural and mechanistic studies illustrating siRNA and miRNA processing, RISC formation, the execution of RNAi by RISC, and the regulation of these pathways, with a specific focus on vertebrate systems.

Adenosine Deaminase↗
PubMed1995

Transcription, splicing and editing of plastid RNAs in the nonphotosynthetic plant Epifagus virginiana.

Expression of the vestigial plastid genome of the nonphotosynthetic, parasitic flowering plant Epifagus virginiana was examined by northern analysis and by characterization of cDNAs. Probes for each of 12 plastid genes tested hybridized to all lanes of northern blots containing total RNA prepared from stems and fruits of Epifagus and from leaves of tobacco. Certain transcript patterns in Epifagus plastids are highly complex and similar to those of tobacco operons. In contrast, genes such as rps2, which have become orphaned in Epifagus as a result of evolutionary loss of formerly cotranscribed genes, show simpler transcript patterns in Epifagus than in tobacco. Sizing and sequencing of cDNAs generated by reverse transcriptase-PCR for three genes, rps12, rpl2, and clpP, show that their transcripts are properly cis- and/or trans-spliced at the same five group II intron insertion sites used in photosynthetic plants. A single, conventional C-->U edit in rps12 was found among the total of 1401 nucleotides of cDNA sequence that was determined for the three genes. An octanucleotide sequence identical to a putative guide RNA of plant organelles and perfectly complementary to the rps12 edit site itself was identified just 200 bp upstream of the edit site. These data, together with previous results from the complete sequencing of the Epifagus plastid genome, provide compelling evidence that this degenerate genome is nonetheless expressed and functional. Analysis of the putative maturase MatK, encoded by the group II intron of trnK in photosynthetic land plants but by a freestanding gene in Epifagus, leads us to hypothesize that it acts 'in trans' to assist the splicing of group II introns other than the one in which it is normally encoded.

Adenosine Triphosphatases↗
PubMed2025

Small RNAs derived from avocado sunblotch viroid and their association with bleaching symptoms: implications for pathogenesis in avocado sunblotch disease.

Avocado sunblotch viroid (ASBVd) is a structured RNA molecule responsible for sunblotch disease of avocado, characterised by distinct chloroses of fruit, leaves, and stems. Despite its impact on avocado, the mechanism by which ASBVd elicits sunblotch symptoms remains unknown. Previous studies on other avsunviroids have shown that viroid-derived small RNAs (vd-sRNAs) with specific sequence mutations can trigger leaf chlorosis via RNA silencing of host genes. Building on this knowledge, we aimed to shed light on the molecular basis of ASBVd pathogenesis by analysing ASBVd sequence variants and ASBVd-sRNAs from bleached and asymptomatic leaf tissues of sunblotch-affected avocado trees. Sequencing of ASBVd clones revealed that variants carrying the pathogenic determinant for bleaching were present in both green and yellow leaf tissues. Next-generation sequencing (NGS) identified ASBVd-sRNAs that varied in abundance between symptomatic and asymptomatic leaf tissues, correlating with viroid titre. We discovered 64 vd-sRNAs spanning the pathogenic region of the ASBVd genome, which were almost exclusively found in yellow tissues. The ASBVd-sRNAs containing the bleaching-associated mutation were predicted to target numerous avocado transcripts for degradation, with 25 of these transcripts significantly downregulated in bleached tissues. Notably, one of these genes, encoding a chloroplastic protein, demonstrated strong evidence of ASBVd-sRNA-guided RNA silencing, presenting a promising candidate for future research into the molecular trigger for ASBVd-induced bleaching symptoms. This study is the first to investigate ASBVd-sRNAs in bleached leaves using NGS. Our findings support the role of RNA silencing in sunblotch symptom development and reveal a unique silencing trigger compared to other avsunviroids.

Persea↗
PubMed2006

Crystal structure determination and site-directed mutagenesis of the Pyrococcus abyssi aCBF5-aNOP10 complex reveal crucial roles of the C-terminal domains of both proteins in H/ACA sRNP activity.

In archaeal rRNAs, the isomerization of uridine into pseudouridine (Psi) is achieved by the H/ACA sRNPs and the minimal set of proteins required for RNA:Psi-synthase activity is the aCBF5-aNOP10 protein pair. The crystal structure of the aCBF5-aNOP10 heterodimer from Pyrococcus abyssi was solved at 2.1 A resolution. In this structure, protein aNOP10 has an extended shape, with a zinc-binding motif at the N-terminus and an alpha-helix at the C-terminus. Both motifs contact the aCBF5 catalytic domain. Although less efficiently as does the full-length aNOP10, the aNOP10 C-terminal domain binds aCBF5 and stimulates the RNA-guided activity. We show that the C-terminal domain of aCBF5 (the PUA domain), which is wrapped by an N-terminal extension of aCBF5, plays a crucial role for aCBF5 binding to the guide sRNA. Addition of this domain in trans partially complement particles assembled with an aCBF5DeltaPUA truncated protein. In the crystal structure, the aCBF5-aNOP10 complex forms two kinds of heterotetramers with parallel and perpendicular orientations of the aNOP10 terminal alpha-helices, respectively. By gel filtration assay, we showed that aNOP10 can dimerize in solution. As both residues Y41 and L48 were needed for dimerization, the dimerization likely takes place by interaction of parallel alpha-helices.

Amino Acid Sequence↗
PubMed2004

Recent applications of RNAi in mammalian systems.

RNAi is a powerful cellular mechanism that involves targeted destruction of mRNAs. Although the phenomenon was first discovered in plants and lower eukaryotic organisms, it was later discovered as an important genetic regulatory mechanism in mammalian cells. RNAi is triggered by double stranded RNAs that are cleaved into short 21-23 base pair duplexes by an RNAse III type enzyme called Dicer. The short RNAs, termed small interfering RNAs (siRNAs), act as triggers for targeted RNA degradation. One of the two strands is selectively incorporated into a complex of proteins called the RNA induced silencing complex, or RISC. The incorporated small RNA guides the complex to the complementary target sequence, and this event is followed by endonucleolytic cleavage of the target and recycling of RISC. In mammalian cells, siRNAs do not activate interferon pathway genes, thereby making these powerful tools for sequence specific knockdown of RNAs. In this article we review the methods for programming mammalian cells with siRNAs, and overview a number of applications ranging from targeting oncogenes to inhibiting viral replication. The article also summarizes some important biological conclusions that can be drawn from selective downregulation of certain mRNA targets and addresses potential uses of RNAi as a new therapeutic modality.

Animals↗
PubMed2004

Biochemical identification of Argonaute 2 as the sole protein required for RNA-induced silencing complex activity.

RNA interference is carried out by the small double-stranded RNA-induced silencing complex (RISC). The RISC-bound small RNA guides the RISC complex to identify and cleave mRNAs with complementary sequences. The proteins that make up the RISC complex and cleave mRNA have not been unequivocally defined. Here, we report the biochemical purification of RISC activity to homogeneity from Drosophila Schnieder 2 cell extracts. Argonaute 2 (Ago-2) is the sole protein component present in the purified, functional RISC. By using a bioinformatics method that combines sequence-profile analysis with predicted protein secondary structure, we found homology between the PIWI domain of Ago-2 and endonuclease V and identified potential active-site amino acid residues within the PIWI domain of Ago-2.

Amino Acid Sequence↗
PubMed2005

Antiviral silencing in animals.

RNA silencing or RNA interference (RNAi) refers to the small RNA-guided gene silencing mechanism conserved in a wide range of eukaryotic organisms from plants to mammals. As part of this special issue on the biology, mechanisms and applications of RNAi, here we review the recent advances on defining a role of RNAi in the responses of invertebrate and vertebrate animals to virus infection. Approximately 40 miRNAs and 10 RNAi suppressors encoded by diverse mammalian viruses have been identified. Assays used for the identification of viral suppressors and possible biological functions of both viral miRNAs and suppressors are discussed. We propose that herpes viral miRNAs may act as specificity factors to initiate heterochromatin assembly of the latent viral DNA genome in the nucleus.

Animals↗
PubMed2006

Formation of the conserved pseudouridine at position 55 in archaeal tRNA.

Pseudouridine (Psi) located at position 55 in tRNA is a nearly universally conserved RNA modification found in all three domains of life. This modification is catalyzed by TruB in bacteria and by Pus4 in eukaryotes, but so far the Psi55 synthase has not been identified in archaea. In this work, we report the ability of two distinct pseudouridine synthases from the hyperthermophilic archaeon Pyrococcus furiosus to specifically modify U55 in tRNA in vitro. These enzymes are (pfu)Cbf5, a protein known to play a role in RNA-guided modification of rRNA, and (pfu)PsuX, a previously uncharacterized enzyme that is not a member of the TruB/Pus4/Cbf5 family of pseudouridine synthases. (pfu)PsuX is hereafter renamed (pfu)Pus10. Both enzymes specifically modify tRNA U55 in vitro but exhibit differences in substrate recognition. In addition, we find that in a heterologous in vivo system, (pfu)Pus10 efficiently complements an Escherichia coli strain deficient in the bacterial Psi55 synthase TruB. These results indicate that it is probable that (pfu)Cbf5 or (pfu)Pus10 (or both) is responsible for the introduction of pseudouridine at U55 in tRNAs in archaea. While we cannot unequivocally assign the function from our results, both possibilities represent unexpected functions of these proteins as discussed herein.

Archaea↗
PubMed2002

Interaction of translation initiation factor eIF4B with the poliovirus internal ribosome entry site.

Poliovirus translation is initiated at the internal ribosome entry site (IRES). Most likely involving the action of standard initiation factors, this highly structured cis element in the 5" noncoding region of the viral RNA guides the ribosome to an internal silent AUG. The actual start codon for viral protein synthesis further downstream is then reached by ribosomal scanning. In this study we show that two of the secondary structure elements of the poliovirus IRES, domain V and, to a minor extent, domain VI, are the determinants for binding of the eukaryotic initiation factor eIF4B. Several mutations in domain V which are known to greatly affect poliovirus growth also seriously impair the binding of eIF4B. The interaction of eIF4B with the IRES is not dependent on the presence of the polypyrimidine tract-binding protein, which also binds to the poliovirus IRES. In contrast to its weak interaction with cellular mRNAs, eIF4B remains tightly associated with the poliovirus IRES during the formation of complete 80S ribosomes. Binding of eIF4B to the IRES is energy dependent, and binding of the small ribosomal subunit to the IRES requires the previous energy-dependent association of initiation factors with the IRES. These results indicate that the interaction of eIF4B with the 3" region of the poliovirus IRES may be directly involved in translation initiation.

5' Untranslated Regions↗
PubMed2026

Synergistic HMGN1 and VP64 Fusions Potentiate High-Precision and PAM-Flexible Base Editing.

RNA-guided CRISPR-derived base editors (BEs) have revolutionized genome editing by enabling targeted base substitutions. However, their application is frequently constrained by the stringent requirement for PAM sequences and low editing precision (bystander editing). Here, we present a robust strategy to overcome these limitations by coupling SpRY, a near-PAM-less Cas9 variant, with truncated CDA1 cytidine deaminases. While this combination enables precise editing of virtually any cytosine in the genome, it initially exhibited suboptimal efficiency. To address this, we systematically screened a diverse panel of candidate DNA-binding proteins and identified that the synergistic fusion of HMGN1 and VP64 substantially enhances editing activity without compromising precision. Importantly, this enhanced editing efficiency was achieved without markedly increasing off-target effects. Our new BEs demonstrated robust performance not only in yeast but also in rice, suggesting broad applicability in gene therapy, precision breeding, and fundamental research.

Gene Editing↗
PubMed1990

An extensively edited mitochondrial transcript in kinetoplastids encodes a protein homologous to ATPase subunit 6.

The mitochondrial MURF4 gene of T. brucei has pronounced G versus C strand bias and heterogeneously sized transcripts, characteristic of genes encoding extensively edited transcripts. We find that MURF4 transcripts of T. brucei are extensively edited throughout by the addition and deletion of numerous uridines, creating potential initiation and termination codons and a continuous open reading frame. A potential guide RNA sequence occurs in a minicircle between inverted repeats. The 5' region of L. tarentolae MURF4 transcripts is also extensively edited, with a created initiation codon. The predicted MURF4 amino acid sequences have homology to those of mitochondrial ATP-ase subunit 6 genes from a variety of organisms. In addition, their hydropathic profiles are quite similar to those of other species. We therefore conclude that MURF4 encodes ATPase subunit 6 genes.

Adenosine Triphosphatases↗