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PubMed1997

Purification of a functional enzymatic editing complex from Trypanosoma brucei mitochondria.

Kinetoplastid mitochondrial RNA editing, the insertion and deletion of U residues, is catalyzed by sequential cleavage, U addition or removal, and ligation reactions and is directed by complementary guide RNAs. We have purified a approximately 20S enzymatic complex from Trypanosoma brucei mitochondria that catalyzes a complete editing reaction in vitro. This complex possesses all four activities predicted to catalyze RNA editing: gRNA-directed endonuclease, terminal uridylyl transferase, 3' U-specific exonuclease, and RNA ligase. However, it does not contain other putative editing complex components: gRNA-independent endonuclease, RNA helicase, endogenous gRNAs or pre-mRNAs, or a 25 kDa gRNA-binding protein. The complex is composed of eight major polypeptides, three of which represent RNA ligase. These findings identify polypeptides representing catalytic editing factors, reveal the nature of this approximately 20S editing complex, and suggest a new model of editosome assembly.

Animals↗

PubMed2001

Annealing of RNA editing substrates facilitated by guide RNA-binding protein gBP21.

RNA editing within the mitochondria of African trypanosomes is characterized by the insertion and deletion of uridylate residues into otherwise incomplete primary transcripts. The reaction takes place in a high molecular mass ribonucleoprotein (RNP) complex of uncertain composition. Furthermore, factors that interact with the RNP complex during the reaction are by and large unknown. Here we present evidence for an editing-related biochemical activity of the gRNA-binding protein gBP21. Using recombinant gBP21 preparations, we show that the protein stimulates the annealing of gRNAs to cognate pre-mRNAs in vitro. This represents the presumed first step of the editing reaction. Kinetic data establish an enhancement of the second order rate constant for the gRNA- pre-mRNA interaction. gBP21-mediated annealing is not exclusive for RNA editing substrates since complementary RNAs, unrelated to the editing process, can also be hybridized. The gBP21-dependent RNA annealing activity was identified in mitochondrial extracts of trypanosomes and can be inhibited by immunoprecipitation of the polypeptide. The data suggest a factor-like contribution of gBP21 to the RNA editing process by accelerating the rate of gRNA-pre-mRNA anchor formation.

Animals↗

PubMed1993

Trypanosoma brucei minicircles encode multiple guide RNAs which can direct editing of extensively overlapping sequences.

Small guide RNAs (gRNAs) may direct RNA editing in kinetoplastid mitochondria. We have characterized multiple gRNA genes from Trypanosoma brucei (EATRO 164), that can specify up to 30% of the editing of the COIII, ND7, ND8, and A6 mRNAs and we have also found that the non-translated region of edited COIII mRNA of strain (EATRO 164) differs from that of another strain. Several of the gRNAs specify overlapping regions of the same mRNA often specifying sequence beyond that required for an anchor duplex with the next gRNA. Some gRNAs have different sequence but specify identical editing of the same region of mRNA. These data indicate a complex gRNA population and consequent complex pattern of editing in T. brucei.

Animals↗

PubMed2001

Cloning and characterization of two guide RNA-binding proteins from mitochondria of Crithidia fasciculata: gBP27, a novel protein, and gBP29, the orthologue of Trypanosoma brucei gBP21.

In kinetoplastid protozoa, mitochondrial (mt) mRNAs are post-transcriptionally edited by insertion and deletion of uridylate residues, the information being provided by guide (g)RNAs. Currently popular mechanisms for the editing process envisage a series of consecutive 'cut-and-paste' reactions, carried out by a complex RNP machinery. Here we report on the purification, cloning and functional analysis of two gRNA-binding proteins of 28.8 (gBP29) and 26.8 kDa (gBP27) from mitochondria of the insect trypanosome Crithidia fasciculata. gBP29 and gBP27 proved to be similar, Arg + Ala-rich proteins, with pI values of approximately 10.0. gBP27 has no homology to known proteins, but gBP29 is the C.fasciculata orthologue of gBP21 from Trypanosoma brucei, a gRNA-binding protein that associates with active RNA editing complexes. As measured in UV cross-linking assays, His-tagged recombinant gBP29 and gBP27 bind to radiolabelled poly(U) and synthetic gRNAs, while competition experiments suggest a role for the gRNA 3'-(U)-tail in binding to these proteins. Immunoprecipitates of mt extracts generated with antibodies against gBP29 also contained gBP27 and vice versa. The immunoprecipitates further harbored a large proportion of the cellular content of four different gRNAs and of edited and pre-edited NADH dehydrogenase subunit 7 mRNAs, but only small amounts of mt rRNAs. In addition, the bulk of gBP29 and gBP27 co-eluted with gRNAs from gel filtration columns in the high molecular weight range. Together, these results suggest that the proteins are part of a large macromolecular complex(es). We infer that gBP29 and gBP27 are components of the C.fasciculata editing machinery that may interact with gRNAs.

Amino Acid Sequence↗

PubMed2000

Complete nucleotide sequence and genome organization of hibiscus chlorotic ringspot virus, a new member of the genus Carmovirus: evidence for the presence and expression of two novel open reading frames.

The complete nucleotide sequence of hibiscus chlorotic ringspot virus (HCRSV) was determined. The genomic RNA (gRNA) is 3,911 nucleotides long and has the potential to encode seven viral proteins in the order of 28 (p28), 23 (p23), 81 (p81), 8 (p8), 9 (p9), 38 (p38), and 25 (p25) kDa. Excluding two unique open reading frames (ORFs) encoding p23 and p25, the ORFs encode proteins with high amino acid similarity to those of carmoviruses. In addition to gRNA, two 3'-coterminated subgenomic RNA (sgRNA) species were identified. Full-length cDNA clones derived from gRNA and sgRNA were constructed under the control of a T7 promoter. Both capped and uncapped transcripts derived from the full-length genomic cDNA clone were infectious. In vitro translation and mutagenesis assays confirmed that all the predicted ORFs except the ORF encoding p8 are translatable, and the two novel ORFs (those encoding p23 and p25) may be functionally indispensable for the viral infection cycle. Based on virion morphology and genome organization, we propose that HCRSV be classified as a new member of the genus Carmovirus in family Tombusviridae.

3' Untranslated Regions↗

PubMed2002

A six-nucleotide segment within the 3' untranslated region of hibiscus chlorotic ringspot virus plays an essential role in translational enhancement.

RNA plant viruses use various translational regulatory mechanisms to control their gene expression. Translational enhancement of viral mRNAs that leads to higher levels of protein synthesis from specific genes may be essential for the virus to successfully compete for cellular translational machinery. The control elements have yet to be analyzed for members of the genus Carmovirus, a small group of plant viruses with positive-sense RNA genomes. In this study, we examined the 3' untranslated region (UTR) of hibiscus chlorotic ringspot virus (HCRSV) genomic RNA (gRNA) and subgenomic RNA (sgRNA) for its role in the translational regulation of viral gene expression. The results showed that the 3' UTR of HCRSV significantly enhanced the translation of several open reading frames on gRNA and sgRNA and a viral gene in a bicistronic construct with an inserted internal ribosome entry site. Through deletion and mutagenesis studies of both the bicistronic construct and full-length gRNA, we demonstrated that a six-nucleotide sequence, GGGCAG, that is complementary to the 3' region of the 18S rRNA and a minimal length of 180 nucleotides are required for the enhancement of translation induced by the 3' UTR.

3' Untranslated Regions↗

PubMed2000

Trypanosoma brucei guide RNA poly(U) tail formation is stabilized by cognate mRNA.

Guide RNAs (gRNAs) are small RNAs that provide specificity for uridine addition and deletion during mRNA editing in trypanosomes. Terminal uridylyl transferase (TUTase) adds uridines to pre-mRNAs during RNA editing and adds a poly(U) tail to the 3' end of gRNAs. The poly(U) tail may stabilize the association of gRNAs with cognate mRNA during editing. Both TUTase and gRNAs associate with two ribonucleoprotein complexes, I (19S) and II (35S to 40S). Complex II is believed to be the fully assembled active editing complex, since it contains pre-edited mRNA and enzymes thought necessary for editing. Purification of TUTase from mitochondrial extracts resulted in the identification of two chromatographically distinct TUTase activities. Stable single-uridine addition to different substrate RNAs is performed by the 19S complex, despite the presence of a uridine-specific 3' exonuclease within this complex. Multiple uridines are added to substrate RNAs by a 10S particle that may be an unstable subunit of complex I lacking the uridine-specific 3' exonuclease. Multiple uridines could be stably added onto gRNAs by complex I when the cognate mRNA is present. We propose a model in which the purine-rich region of the cognate mRNA protects the uridine tail from a uridine exonuclease activity that is present within the complex. To test this model, we have mutated the purine-rich region of the pre-mRNA to abolish base-pairing interaction with the poly(U) tail of the gRNA. This RNA fails to protect the uridine tail of the gRNA from exoribonucleolytic trimming and is consistent with a role for the purine-rich region of the mRNA in gRNA maturation.

Animals↗

PubMed2005

The zebrafish progranulin gene family and antisense transcripts.

BACKGROUND: Progranulin is an epithelial tissue growth factor (also known as proepithelin, acrogranin and PC-cell-derived growth factor) that has been implicated in development, wound healing and in the progression of many cancers. The single mammalian progranulin gene encodes a glycoprotein precursor consisting of seven and one half tandemly repeated non-identical copies of the cystine-rich granulin motif. A genome-wide duplication event hypothesized to have occurred at the base of the teleost radiation predicts that mammalian progranulin may be represented by two co-orthologues in zebrafish. RESULTS: The cDNAs encoding two zebrafish granulin precursors, progranulins-A and -B, were characterized and found to contain 10 and 9 copies of the granulin motif respectively. The cDNAs and genes encoding the two forms of granulin, progranulins-1 and -2, were also cloned and sequenced. Both latter peptides were found to be encoded by precursors with a simplified architecture consisting of one and one half copies of the granulin motif. A cDNA encoding a chimeric progranulin which likely arises through the mechanism of trans-splicing between grn1 and grn2 was also characterized. A non-coding RNA gene with antisense complementarity to both grn1 and grn2 was identified which may have functional implications with respect to gene dosage, as well as in restricting the formation of the chimeric form of progranulin. Chromosomal localization of the four progranulin (grn) genes reveals syntenic conservation for grna only, suggesting that it is the true orthologue of mammalian grn. RT-PCR and whole-mount in situ hybridization analysis of zebrafish grns during development reveals that combined expression of grna and grnb, but not grn1 and grn2, recapitulate many of the expression patterns observed for the murine counterpart. This includes maternal deposition, widespread central nervous system distribution and specific localization within the epithelial compartments of various organs. CONCLUSION: In support of the duplication-degeneration-complementation model of duplicate gene retention, partitioning of expression between grna and grnb was observed in the intermediate cell mass and yolk syncytial layer, respectively. Taken together these expression patterns suggest that the function of an ancestral grn gene has been devolved upon four paralogues in zebrafish.

Amino Acid Motifs↗

PubMed2004

The 3'-untranslated region of cytochrome oxidase II mRNA functions in RNA editing of African trypanosomes exclusively as a cis guide RNA.

RNA editing in trypanosomes is a post-transcriptional process responsible for correcting the coding sequences of many mitochondrial mRNAs. Uridines are specifically added or deleted from mRNA by an enzymatic cascade in which a pre-edited mRNA is specifically cleaved, uridines are added or removed, and the corrected mRNA is ligated. The process is directed by RNA molecules, termed guide RNAs (gRNA). The ability of this class of small, noncoding RNA to function in RNA editing is essential for these organisms. Typically, gRNAs are transcribed independent of the their cognate mRNA and anneal to form a binary RNA complex . An exception for this process may be cytochrome oxidase subunit II (COII) mRNA since a gene encoding a trans acting gRNA has not been identified. Using an in vitro editing assay we find that the 3' UTR of COII, indeed, functions as a guide for both the site and number of uridines added to the coding region of the COII mRNA. We further show that the guiding sequence within the COII 3' UTR can only function in COII editing when contiguous with the editing substrate, indicating that the 3' UTR of COII lacks sequence or structure information necessary to function as a trans-acting gRNA. While other RNAs have been shown to "guide" RNA processing reactions, our discovery that the COII 3' UTR directs editing of its cognate mRNA in cis, is a unique function for a 3' UTR. The findings described here have led us to propose a new model for the evolution of gRNAs in kinetoplastids.

3' Untranslated Regions↗

PubMed1995

In vitro RNA editing-like activity in a mitochondrial extract from Leishmania tarentolae.

A mitochondrial extract from Leishmania tarentolae directs the incorporation of uridylate (U) residues within the pre-edited domain of synthetic cytochrome b (CYb) and NADH dehydrogenase subunit 7 mRNA. This has several characteristics of an in vitro RNA editing activity, but no direct evidence for involvement of guide RNAs was obtained. Inhibition by micrococcal nuclease suggests a requirement for some type of endogenous RNA. The limitation of internal U-incorporation to the pre-edited region in the CYb mRNA and the inhibition by deletion or substitution of both mRNA anchor sequences for CYb gRNA-I and -II could be consistent either with a gRNA-mediated process or a secondary structure-mediated process. A low level of incorporation of [alpha-32P]CTP occurs at the same sites as UTP. Internal U-incorporation activity is selectively inhibited by heterologous RNAs, suggesting an involvement of low affinity RNA-binding proteins which can be competed by the added RNA.

Animals↗

PubMed2025

Performance of Subgenomic RT-PCR for Predicting SARS-CoV-2 Infectivity Compared to Genomic RT-PCR and Culture Isolation.

SARS-CoV-2 clinical samples can be detected as positive for a long period of time using real-time RT-PCR, even when patients are no longer infectious. Viral culture is the gold standard for assessing a patient's infectivity, but it is a time-consuming technique and lacks sensitivity. SARS-CoV-2 subgenomic RNA (sgRNA) detection has been used as a proxy for assessing the infectivity but only a limited number of studies have described its use in vitro and in clinical samples. This study aimed to evaluate the correlation between results from viral culture, genomic RT-PCR (gRT-PCR), and subgenomic RT-PCR (sgRT-PCR) during in vitro infection and in clinical samples. In vitro viral replication kinetics showed that both genomic RNA (gRNA) and subgenomic RNA (sgRNA) levels remained stable up to 21 days in the absence of replication-competent virus. Using clinical samples, sgRNA was detected in 87.5% of culture-positive samples, demonstrating better performances than gRT-PCR (Positive predictive value (PPV) 93.3% and Negative predictive value (NPV) of 87.5%) and an almost perfect agreement with culture results (Cohen κ = 0.81 [95% CI: 0.66-0.95]). These findings suggest that testing for sgRNA and/or using a gRNA Ct cut-off of 21.2 could be used as a proxy to determine the presence of SARS-CoV-2 replication-competent virus.

Humans↗

PubMed2001

The nucleotide sequence and genomic organization of Citrus leaf blotch virus: candidate type species for a new virus genus.

The complete nucleotide sequence of Citrus leaf blotch virus (CLBV) was determined. CLBV genomic RNA (gRNA) has 8747 nt, excluding the 3'-terminal poly(A) tail, and contains three open reading frames (ORFs) and untranslated regions (UTR) of 73 and 541 nucleotides at the 5' and 3' termini, respectively. ORF1 potentially encodes a 227.4-kDa polypeptide, which has methyltransferase, papain-like protease, helicase, and RNA-dependent RNA polymerase motifs. ORF2 encodes a 40.2-kDa polypeptide containing a motif characteristic of cell-to-cell movement proteins. The 40.7-kDa polypeptide encoded by ORF3 was identified as the coat protein. The genome organization of CLBV resembles that of viruses in the genus Trichovirus, but they differ in various aspects: (i) in trichoviruses ORF2 overlaps ORFs 1 and 3, whereas in CLBV, ORFs 2 and 3 are separated and ORFs 1 and 2 overlap in one nucleotide; (ii) CLBV gRNA and CP are larger than those of trichoviruses; and (iii) the CLBV 3' UTR is larger than that of trichoviruses. Phylogenetic comparisons based on CP amino acid signatures clearly separates CLBV from trichoviruses. Also contrasting with trichoviruses, CLBV could not be transmitted to Chenopodium quinoa Willd. Considering these singularities, we propose that CLBV should be included in a new virus genus.

Amino Acid Sequence↗

PubMed2027

Systematic Dissection of Key Driver Perturbation Signatures in Single Cells via ECCITE-seq.

CRISPR screens, such as expanded CRISPR-compatible cellular indexing of transcriptomes and epitopes by sequencing (ECCITE-seq), enable the simultaneous measurement of transcriptomes, gRNA identity, and cell-surface protein expression at single-cell resolution to systematically interrogate gene function. This platform provides a powerful and scalable experimental approach for validating disease-associated regulators identified by large-scale association studies and other computational methods, including network-based analyses of multi-omics data. Here, as an example application, we describe an ECCITE-seq framework to characterize the transcriptomic consequences of perturbing multiple neuronal key driver genes associated with Alzheimer's disease (AD) in human-induced pluripotent stem cell (hiPSC)-derived neurons. More broadly, by integrating customized pooled gRNA libraries with different CRISPR effectors across multiple cell types, this approach allows for the assessment of the regulatory impact of candidate genes implicated in development and disease processes.

Humans↗

PubMed1993

Analysis of messenger RNA within virions of IBV.

The presence of subgenomic mRNAs (sgRNAs) in virions of infectious bronchitis virus was examined by probing Northern blots of RNA extracted from virions using as a probe a cDNA of the 3'-terminal nucleocapsid protein (N) gene. The sgRNAs were readily detected even after extensive purification of virions and after RNase A treatment of virions. The molar ratio of gRNA to each sgRNA was in the range 25 to 400 for IBV-M41 and 10 to 30 for IBV-Beaudette. After comparison with the molar ratios of genomic to intracellular viral sgRNAs it was estimated that the efficiency of incorporation of gRNA into virions was approximately 100 to 500-fold greater than for sgRNAs in the case of M41 and 20 to 100-fold for Beaudette, depending on the sgRNA species. It is concluded that sgRNAs can be present within IBV virions. Approximately 1 in 3 Beaudette virions and 1 in 20 M41 particles might contain a single copy of one sgRNA.

Animals↗

PubMed1995

In vivo testing of a tobacco plastid DNA segment for guide RNA function in psbL editing.

A C-to-U RNA editing event creates a functional initiation codon for translation of the psbL mRNA in tobacco plastids. Small trans-acting guide RNAs (gRNAs) have been shown to be involved in editing site selection in kinetoplastid mitochondria. A computer search of the tobacco plastid genome (ptDNA) identified such a putative gRNA, a 14-nucleotide sequence motif that is complementary to the psbL mRNA, including the A nucleotide required to direct the C-to-U change. The critical A nucleotide of the putative gRNA gene was changed to G by plastid transformation. We report here that the introduced mutation did not abolish psbL editing. Since no other region of the plastid genome contains significant complementarity to the psbL editing site we suggest that, if gRNAs serve as trans-acting factors for plastid psbL mRNA editing, they either have only a limited complementarity to the editing site, or are encoded in the nuclear genome.

Base Sequence↗

PubMed2025

Engineered Lactiplantibacillus plantarum and Levilactobacillus brevis utilizing ribonucleoprotein-mediated editing for inactivation of hemolysin gene.

Lactiplantibacillus plantarum and Levilactobacillus brevis are widely used probiotics with significant potential as chassis organisms for probiotic engineering. However, their bioengineering remains underdeveloped compared to that of other probiotic bacteria due to the limited availability of genetic tools. Although CRISPR-Cas systems have shown promise for genome editing in Lactobacillus species, strain- or site-specific targeting challenges must be overcome to enhance their broader applicability. This study aimed to develop a novel editing system with reduced dependency on plasmids and antibiotics in L. plantarum WCFS1, L. plantarum SPC 72 - 1 and L. brevis SPC-SNU 70 - 2 using a Cas9-gRNA ribonucleoprotein (RNP) complex. Although the hlyIII gene has been annotated as a hemolysin-related gene in several Lactobacillus genomes, no functional hemolytic activity has been definitively demonstrated to date. In this study, hlyIII was selected as a target to evaluate genome editing efficiency and to assess its potential relevance to strain safety. To construct ΔhlyIII strains, the RNP complex targeting hlyIII was separately transformed with recombinase RecE/T and double-stranded donor DNA. As a result, ΔhlyIII mutants were obtained under optimized electroporation conditions. Sequencing analysis revealed a 50 bp deletion and the introduction of a stop codon in hlyIII across all mutant strains. The hemolytic activity test showed a reduction in free hemoglobin levels in the ΔhlyIII strains compared to the wild type: 27.0%, 74.3%, and 5.0% in L. plantarum WCFS1, L. plantarum SPC 72 - 1, and L. brevis SPC-SNU 70 - 2, respectively. These results suggest strain-dependent differences in hemolytic activity and indicate that inactivation of hlyIII may contribute to reduced hemolysis, although further validation is needed to clarify its functional role. In conclusion, the hlyIII gene was successfully edited in L. plantarum and L. brevis using Cas9-gRNA ribonucleoprotein-mediated editing, demonstrating the feasibility of this genome editing platform for application in probiotic strains.

Gene Editing↗

PubMed1990

Guide RNAs in kinetoplastid mitochondria have a nonencoded 3' oligo(U) tail involved in recognition of the preedited region.

Maxicircle-encoded guide RNAs (gRNAs) for cytochrome b and maxicircle unidentified reading frames 2 and 3 (MURF2 and MURF3) were isolated by hybrid selection and sequenced. All three gRNAs contained nonencoded 3' oligo(U) tails 5-24 nucleotides in length, with a mean length of approximately 15 nucleotides. Secondary structure calculations indicate a functional role of the 3' oligo(U) tail in stabilizing the initial hybrid formed between the gRNA and the preedited mRNA, and allowed the identification of potential mRNA recognition sites for an editing complex. In addition, isolated MURF2 gRNA-II could be 5' capped with [alpha-32P]-GTP and guanylyltransferase, suggesting that at least some gRNAs represent primary transcripts.

Animals↗

PubMed2006

Crystal structures of T. brucei MRP1/MRP2 guide-RNA binding complex reveal RNA matchmaking mechanism.

The mitochondrial RNA binding proteins MRP1 and MRP2 form a heteromeric complex that functions in kinetoplastid RNA editing. In this process, MRP1/MRP2 serves as a matchmaker by binding to guide RNAs and facilitating their hybridization with cognate preedited mRNAs. To understand the mechanism by which this complex performs RNA matchmaking, we determined structures of Trypanosoma brucei apoMRP1/MRP2 and an MRP1/MRP2-gRNA complex. The structures show that MRP1/MRP2 is a heterotetramer and, despite little sequence homology, each MRP subunit exhibits the same "Whirly" transcription-factor fold. The gRNA molecule binds to the highly basic beta sheet surface of the MRP complex via nonspecific, electrostatic contacts. Strikingly, while the gRNA stem/loop II base is anchored to the basic surface, stem/loop I (the anchor sequence) is unfolded and its bases exposed to solvent. Thus, MRP1/MRP2 acts as an RNA matchmaker by stabilizing the RNA molecule in an unfolded conformation suitable for RNA-RNA hybridization.

Amino Acid Sequence↗