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At least 793 records · Page 44Linked to original sources
PubMed1998

Three small nucleolar RNAs identified from the spliced leader-associated RNA locus in kinetoplastid protozoans.

First characterized in Trypanosoma brucei, the spliced leader-associated (SLA) RNA gene locus has now been isolated from the kinetoplastids Leishmania tarentolae and Trypanosoma cruzi. In addition to the T. brucei SLA RNA, both L. tarentolae and T. cruzi SLA RNA repeat units also yield RNAs of 75 or 76 nucleotides (nt), 92 or 94 nt, and approximately 450 or approximately 350 nt, respectively, each with significant sequence identity to transcripts previously described from the T. brucei SLA RNA locus. Cell fractionation studies localize the three additional RNAs to the nucleolus; the presence of box C/D-like elements in two of the transcripts suggests that they are members of a class of small nucleolar RNAs (snoRNAs) that guide modification and cleavage of rRNAs. Candidate rRNA-snoRNA interactions can be found for one domain in each of the C/D element-containing RNAs. The putative target site for the 75/76-nt RNA is a highly conserved portion of the small subunit rRNA that contains 2'-O-ribose methylation at a conserved position (Gm1830) in L. tarentolae and in vertebrates. The 92/94-nt RNA has the potential to form base pairs near a conserved methylation site in the large subunit rRNA, which corresponds to position Gm4141 of small rRNA 2 in T. brucei. These data suggest that trypanosomatids do not obey the general 5-bp rule for snoRNA-mediated methylation.

Animals↗
PubMed1992

Chimeric and truncated RNAs in Trypanosoma brucei suggest transesterifications at non-consecutive sites during RNA editing.

RNA editing adds and removes uridines at specific sites in several mitochondrial transcripts in kinetoplastid parasites probably as specified by guide RNAs (gRNAs) that are complementary to the final edited sequence. Editing has been postulated to involve transesterification which predicts (1) chimeric molecules with a gRNA covalently attached by its non-encoded oligo U tail to an internal editing site in the mRNA and (2) the corresponding truncated 5' portions of the mRNAs. We have characterized cDNAs representing a large number of both types of intermediates from Trypanosoma brucei. The lengths of both U tails and encoded gRNA sequences vary greatly in length. The majority of encoded gRNA sequences are shorter than predicted based on their minicircle coding sequences. Analysis of the predominant sites of gRNA attachment in chimeras suggests that the transesterifications that religate the truncated 5' mRNAs may proceed more rapidly at editing sites at the 5' end of an editing domain and at sites of U deletion. Partially edited sequences in the mRNA portion of chimeras and at the 3' ends of truncated 5' mRNAs also indicate a non-consecutive order of site selection during RNA editing.

Animals↗
PubMed2002

Cajal body-specific small nuclear RNAs: a novel class of 2'-O-methylation and pseudouridylation guide RNAs.

Cajal (coiled) bodies are conserved subnuclear organelles that are present in the nucleoplasm of both animal and plant cells. Although Cajal bodies were first described nearly 100 years ago, their function has remained largely speculative. Here, we describe a novel class of human small nuclear RNAs that localize specifically to Cajal bodies. The small Cajal body-specific RNAs (scaRNAs) are predicted or have already been demonstrated to function as guide RNAs in site-specific synthesis of 2'-O-ribose-methylated nucleotides and pseudouridines in the RNA polymerase II-transcribed U1, U2, U4 and U5 spliceosomal small nuclear RNAs (snRNAs). Our results provide strong support for the idea that the Cajal body, this mysterious nuclear organelle, provides the cellular locale for post-transcriptional modification of spliceosomal snRNAs.

Animals↗
PubMed1992

Kinetoplastid RNA editing: in vitro formation of cytochrome b gRNA-mRNA chimeras from synthetic substrate RNAs.

RNA editing in the kinetoplastid Trypanosoma brucei results in the addition and deletion of uridine residues within several mitochondrial mRNAs. The site and number of uridines added appears to be directed by small (approximately 70 nt) guide RNAs (gRNAs), which can base pair to the edited sequences. We examined reactions involving synthetic cytochrome b (CYb) gRNA and pre-edited mRNA in vitro. A major product of the in vitro reaction is a chimeric RNA molecule containing both gRNA and mRNA sequences. Formation of the CYb gRNA-mRNA chimera was specific, since such molecules did not accumulate when either the gRNA or mRNA was substituted with control RNAs. The reaction required a free 3' hydroxyl on the gRNA and was unaffected by capping of the gRNA's 5' end. Direct RNA sequencing indicated that the CYb gRNA is covalently linked via its 3' poly(U) tail to one of the editing sites on the CYb mRNA. These results suggest that the U's added during editing are donated by the poly(U) tail of a gRNA via a chimeric gRNA-mRNA intermediate.

Animals↗
PubMed2002

Prevention of HIV-1 infection in human peripheral blood mononuclear cells by specific RNA interference.

The RNA interference (RNAi) phenomenon is a recently observed process in which the introduction of a double-stranded RNA (dsRNA) into a cell causes the specific degradation of a mRNA containing the same sequence. The 21-23 nt guide RNAs, generated by RNase III cleavage from longer dsRNAs, are associated with sequence-specific mRNA degradation. Here, we show that dsRNA specifically suppresses the expression of HIV-1 genes. To study dsRNA-mediated gene interference in HIV-1-infected cells, we have designed six long dsRNAs containing the HIV-1 gag and env genes. HIV-1 replication was totally suppressed in a sequence-specific manner by the dsRNAs in HIV-1-infected cells. Especially, E2 dsRNA containing the major CD4-binding domain sequence of gp120, as the target of the HIV-1 env gene, dramatically inhibited the expression of the HIV-1 p24 antigen in PBMCs for a relatively long time. The dsRNA interference method seems to be a promising new strategy for anti-HIV-1 gene therapeutics.

Animals↗
PubMed2005

Minimal pre-mRNA substrates with natural and converted sites for full-round U insertion and U deletion RNA editing in trypanosomes.

Trypanosome RNA editing by uridylate insertion or deletion cycles is a mitochondrial mRNA maturation process catalyzed by multisubunit complexes. A full-round of editing entails three consecutive steps directed by partially complementary guide RNAs: pre-mRNA cleavage, U addition or removal, and ligation. The structural and functional composition of editing complexes is intensively studied, but their molecular interactions in and around editing sites are not completely understood. In this study, we performed a systematic analysis of distal RNA requirements for full-round insertion and deletion by purified editosomes. We define minimal substrates for efficient editing of A6 and CYb model transcripts, and established a new substrate, RPS12. Important differences were observed in the composition of substrates for insertion and deletion. Furthermore, we also showed for the first time that natural sites can be artificially converted in both directions: from deletion to insertion or from insertion to deletion. Our site conversions enabled a direct comparison of the two editing kinds at common sites during substrate minimization and demonstrate that all basic determinants directing the editosome to carry out full-round insertion or deletion reside within each editing site. Surprisingly, we were able to engineer a deletion site into CYb, which exclusively undergoes insertion in nature.

Adenosine Triphosphatases↗
PubMed2005

RNA silencing systems and their relevance to plant development.

RNA silencing refers to a broad range of phenomena sharing the common feature that large, double-stranded RNAs or stem-loop precursors are processed to ca. 21-26 nucleotide small RNAs, which then guide the cleavage of cognate RNAs, block productive translation of these RNAs, or induce methylation of specific target DNAs. Although the core mechanisms are evolutionarily conserved, epigenetic maintenance of silencing by amplification of small RNAs and the elaboration of mobile, RNA-based silencing signals occur predominantly in plants. Plant RNA silencing systems are organized into a network with shared components and overlapping functions. MicroRNAs, and probably trans-acting small RNAs, help regulate development at the posttranscriptional level. Small interfering RNAs associated with transgene- and virus-induced silencing function primarily in defending against foreign nucleic acids. Another system, which is concerned with RNA-directed methylation of DNA repeats, seems to have roles in epigenetic silencing of certain transposable elements and genes under their control.

DNA Methylation↗
PubMed2006

Mammalian small nucleolar RNAs are mobile genetic elements.

Small nucleolar RNAs (snoRNAs) of the H/ACA box and C/D box categories guide the pseudouridylation and the 2'-O-ribose methylation of ribosomal RNAs by forming short duplexes with their target. Similarly, small Cajal body-specific RNAs (scaRNAs) guide modifications of spliceosomal RNAs. The vast majority of vertebrate sno/scaRNAs are located in introns of genes transcribed by RNA polymerase II and processed by exonucleolytic trimming after splicing. A bioinformatic search for orthologues of human sno/scaRNAs in sequenced mammalian genomes reveals the presence of species- or lineage-specific sno/scaRNA retroposons (sno/scaRTs) characterized by an A-rich tail and an approximately 14-bp target site duplication that corresponds to their insertion site, as determined by interspecific genomic alignments. Three classes of snoRTs are defined based on the extent of intron and exon sequences from the snoRNA parental host gene they contain. SnoRTs frequently insert in gene introns in the sense orientation at genomic hot spots shared with other genetic mobile elements. Previously characterized human snoRNAs are encoded in retroposons whose parental copies can be identified by phylogenic analysis, showing that snoRTs can be faithfully processed. These results identify snoRNAs as a new family of mobile genetic elements. The insertion of new snoRNA copies might constitute a safeguard mechanism by which the biological activity of snoRNAs is maintained in spite of the risk of mutations in the parental copy. I furthermore propose that retroposition followed by genetic drift is a mechanism that increased snoRNA diversity during vertebrate evolution to eventually acquire new RNA-modification functions.

Animals↗
PubMed2005

Transcriptional profile of NeuroD expression in a human fetal astroglial cell line.

NeuroD1, a member of the basic helix-loop-helix (bHLH) protein family, is a transcription factor that plays a pivotal role in terminal differentiation of neural progenitors. The primary objective was to generate an early transcriptional profile triggered by NeuroD1 to guide future studies on mechanisms of neuronal differentiation. The human NeuroD1 coding region was amplified from human fetal brain RNA using specific primers and cloned into a CMV expression vector (CT-GFP-TOPO/pcDNA3.1). Transfection of a fetal glial cell line with this construct resulted in expression of NeuroD1 in 13-15% of the cells. Markers typical of early neuronal development were observed by immunocytochemical staining in a small proportion of transfected cells. To enrich the population of NeuroD1-expressing cells, fluorescence-activated cell sorting (FACS) was used to purify and collect the NeuroD1/GFP+ cells. Total RNA was extracted from the pair of cultures (NeuroD1/GFP vs. control plasmid/GFP) and processed for gene expression studies. A final gene list was composed from those probe sets that were either increased or decreased in the NeuroD1-expressing cells in three independent experiments (p < 0.001). Each gene was investigated further for possible roles in neurogenesis and a subset of 177 genes was chosen based on the following characteristics: a) genes that are potential NeuroD1 dimerization partners, b) genes that modulate other bHLH transcription factors, c) genes related to development, and d) genes associated with neural induction, outgrowth, and terminal differentiation. DNA microarray analysis of NeuroD1 expression in an astroglial cell line produced a "snapshot" transcriptional profile that will be useful in deciphering the complex molecular code that specifies a neuronal fate.

Astrocytes↗
PubMed1998

Origin of the catalytic activity of bovine seminal ribonuclease against double-stranded RNA.

Bovine seminal ribonuclease (RNase) binds, melts, and (in the case of RNA) catalyzes the hydrolysis of double-stranded nucleic acid 30-fold better under physiological conditions than its pancreatic homologue, the well-known RNase A. Reported here are site-directed mutagenesis experiments that identify the sequence determinants of this enhanced catalytic activity. These experiments have been guided in part by experimental reconstructions of ancestral RNases from extinct organisms that were intermediates in the evolution of the RNase superfamily. It is shown that the enhanced interactions between bovine seminal RNase and double-stranded nucleic acid do not arise from the increased number of basic residues carried by the seminal enzyme. Rather, a combination of a dimeric structure and the introduction of two glycine residues at positions 38 and 111 on the periphery of the active site confers the full catalytic activity of bovine seminal RNase against duplex RNA. A structural model is presented to explain these data, the use of evolutionary reconstructions to guide protein engineering experiments is discussed, and a new variant of RNase A, A(Q28L K31C S32C D38G E111G), which contains all of the elements identified in these experiments as being important for duplex activity, is prepared. This is the most powerful catalyst within this subfamily yet observed, some 46-fold more active against duplex RNA than RNase A.

Amino Acid Substitution↗
PubMed2001

Dicer functions in RNA interference and in synthesis of small RNA involved in developmental timing in C. elegans.

Double-stranded RNAs can suppress expression of homologous genes through an evolutionarily conserved process named RNA interference (RNAi) or post-transcriptional gene silencing (PTGS). One mechanism underlying silencing is degradation of target mRNAs by an RNP complex, which contains approximately 22 nt of siRNAs as guides to substrate selection. A bidentate nuclease called Dicer has been implicated as the protein responsible for siRNA production. Here we characterize the Caenorhabditis elegans ortholog of Dicer (K12H4.8; dcr-1) in vivo and in vitro. dcr-1 mutants show a defect in RNAi. Furthermore, a combination of phenotypic abnormalities and RNA analysis suggests a role for dcr-1 in a regulatory pathway comprised of small temporal RNA (let-7) and its target (e.g., lin-41).

Alleles↗
PubMed1998

RNA editing in the free-living bodonid Bodo saltans.

In parasitic kinetoplastid protozoa, mitochondrial (mt) mRNAs are post-transcriptionally edited by insertion and deletion of uridylate residues, the information being provided by guide (g) RNAs. In order to further explore the role and evolutionary history of this process, we searched for editing in mt RNAs of the free-living bodonid Bodo saltans. We found extensive editing in the transcript for NADH dehydrogenase (ND) subunit 5, which is unedited in trypanosomatids. In contrast, B.saltans cytochrome c oxidase (cox) subunit 2 and maxicircle unidentified reading frame (MURF) 2 RNAs display limited editing in the same regions as their trypanosomatid counterparts. A putative intramolecular cox2 gRNA and the gene for gMURF2-I directing the insertion of only one U in the 5' editing domain of MURF2 RNA, are conserved in B.saltans. This lends (further) evolutionary support to the proposed role of these sequences as gRNAs. Phylogenetic analysis showed that B.saltans is more closely related to trypanosomatids than the cryptobiids Trypanoplasma borreli and Cryptobia helicis, in line with the trypanosomatid-like cox2 and MURF2 RNA editing patterns. Nevertheless, other features like the apparent absence of a catenated mtDNA network, are shared with bodonid and cryptobiid species. ND5 RNA editing may represent yet another example of editing 'on the way out' during kinetoplastid evolution, but in view of the fact that cox2 RNA is unedited in T. borreli and C.helicis, we infer that the editing of this RNA may have arisen relatively recently. Our results provide the first examples of RNA editing in a free-living kinetoplastid, indicating that there is no direct link between U-insertion/deletion editing and a parasitic lifestyle.

Amino Acid Sequence↗
PubMed1991

Alternative secondary structures in the 5' exon affect both forward and reverse self-splicing of the Tetrahymena intervening sequence RNA.

The natural splice junction of the Tetrahymena large ribosomal RNA is flanked by hairpins that are phylogenetically conserved. The stem immediately preceding the splice junction involves nucleotides that also base pair with the internal guide sequence of the intervening sequence during splicing. Thus, precursors which contain wild-type exons can form two alternative helices. We have constructed a series of RNAs where the stem-loop in the 5' exon is more or less stable than in the wild-type precursor, and tested them in both forward and reverse self-splicing reactions. The presence of a stable hairpin in ligated exon substrates interferes with the ability of the intervening sequence to integrate at the splice junction. Similarly, the presence of the wild-type hairpin in the 5' exon reduces the rate of splicing 20-fold in short precursors. The data are consistent with a competition between unproductive formation of a hairpin in the 5' exon and productive pairing of the 5' exon with the internal guide sequence. The reduction of splicing by a hairpin that is a normal feature of rRNA structure is surprising; we propose that this attenuation is relieved in the natural splicing environment.

Animals↗
PubMed2006

The nucleolar protein Esf2 interacts directly with the DExD/H box RNA helicase, Dbp8, to stimulate ATP hydrolysis.

While 18 putative RNA helicases are involved in ribosome biogenesis in Saccharomyces cerevisiae, their enzymatic properties have remained largely biochemically uncharacterized. To better understand their function, we examined the enzymatic properties of Dpb8, a DExD/H box protein previously shown to be required for the synthesis of the 18S rRNA. As expected for an RNA helicase, we demonstrate that recombinant Dbp8 has ATPase activity in vitro, and that this activity is dependent on an intact ATPase domain. Strikingly, we identify Esf2, a nucleolar putative RNA binding protein, as a binding partner for Dbp8, and show that it enhances Dbp8 ATPase activity by decreasing the K(M) for ATP. Thus, we have uncovered Esf2 as the first example of a protein co-factor that has a stimulatory effect on a nucleolar RNA helicase. We show that Esf2 can bind to pre-rRNAs and speculate that it may function to bring Dbp8 to the pre-rRNA, thereby both regulating its enzymatic activity and guiding Dbp8 to its site of action.

Adenosine Triphosphatases↗
PubMedDate not supplied

Preoperative diagnosis of suspicious parathyroid adenomas by RT-PCR using mRNA extracted from leftover cells in a needle used for ultrasonically guided fine needle aspiration cytology.

OBJECTIVE: To determine the usefulness of reverse transcriptase-polymerase chain reaction (RT-PCR) detection of parathormone (PTH) gene mRNA in needle aspirates to confirm the parathyroid nature of suspicious cervical lesions in patients with hyperparathyroidism. STUDY DESIGN: Ultrasound-guided fine needle aspiration cytology (FNAC) was performed on 12 patients with suspected parathyroid adenomas. The aspirates were subjected to cytologic and chromogranin A examination. Messenger RNA was isolated from left-over cells within the needle, and RT-PCR was used to amplify mRNA encoding PTH. RESULTS: The 12 aspirates were positive by PTH RT-PCR, and molecular diagnosis was confirmed by histologic examination. The lower sensitivity of cytologic and immunocytochemical methods was demonstrated, respectively, by 67% and 50% of positive results. Sensitivity assays demonstrated that with our system, PTH RT-PCR products are obtained from as few as 10 pg of parathyroid mRNA. CONCLUSION: The present study showed that RT-PCR-based analysis of PTH gene transcripts in aspirates, obtained by US-guided FNAC of cervical lesions suspicious for parathyroid adenoma, is a feasible, sensitive and specific method for preoperative diagnosis of parathyroid adenomas.

Adenoma↗
PubMed1994

Elimination of hepatitis delta virus infection after loss of hepatitis B surface antigen in patients with chronic delta hepatitis.

The aim of this study was to evaluate whether patients with chronic hepatitis delta virus (HDV) infection treated with alpha interferon and subsequent loss of hepatitis B surface antigen (HBsAg) eliminate HDV. HDV RNA was detected in 26 of 28 patients with chronic delta hepatitis using the polymerase chain reaction. Seventeen patients in whom HDV RNA was detected were treated with alpha interferon; in 65%, HDV RNA remained detectable during treatment or reappeared after stopping therapy whereas in three patients HDV RNA remained absent (17.5%). HDV RNA became and remained undetectable in serum and liver of two of these three patients who lost HBsAg from serum and in one patient who was intermittently HBsAg negative during therapy. After loss of HBsAg, hepatitis B virus (HBV) DNA was still detectable in the liver, but not HBV RNA, indicating absent or very low HBV replication. Three patients were lost to follow up (17.5%). Two nontreated patients with chronic HDV infection also lost HBsAg during follow up; HDV RNA also became undetectable in their serum. Thus, HDV replication does not persist after the loss of HBsAg. Clearance of HBsAg may be a useful guide to when therapy can be stopped.

Adolescent↗
PubMed2002

RGM is a repulsive guidance molecule for retinal axons.

Axons rely on guidance cues to reach remote targets during nervous system development. A well-studied model system for axon guidance is the retinotectal projection. The retina can be divided into halves; the nasal half, next to the nose, and the temporal half. A subset of retinal axons, those from the temporal half, is guided by repulsive cues expressed in a graded fashion in the optic tectum, part of the midbrain. Here we report the cloning and functional characterization of a membrane-associated glycoprotein, which we call RGM (repulsive guidance molecule). This molecule shares no sequence homology with known guidance cues, and its messenger RNA is distributed in a gradient with increasing concentration from the anterior to posterior pole of the embryonic tectum. Recombinant RGM at low nanomolar concentration induces collapse of temporal but not of nasal growth cones and guides temporal retinal axons in vitro, demonstrating its repulsive and axon-specific guiding activity.

Amino Acid Sequence↗