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PubMed2003

An uncapped RNA suggests a model for Caenorhabditis elegans polycistronic pre-mRNA processing.

Polycistronic pre-mRNAs from Caenohabditis elegans operons are processed by internal cleavage and polyadenylation to create 3' ends of mature mRNAs. This is accompanied by trans-splicing with SL2 approximately 100 nucleotides downstream of the 3' end formation sites to create the 5' ends of downstream mRNAs. SL2 trans-splicing depends on a U-rich element (Ur), located approximately 70 nucleotides upstream of the trans-splice site in the intercistronic region (ICR), as well as a functional 3' end formation signal. Here we report the existence of a novel gene-length RNA, the Ur-RNA, starting just upstream of the Ur element. The expression of Ur-RNA is dependent on 3' end formation as well as on the presence of the Ur element, but does not require a trans-splice site. The Ur-RNA is not capped, and alteration of the location of the Ur element in either the 5' or 3' direction alters the location of the 5' end of the Ur-RNA. We propose that a 5' to 3' exonuclease degrades the precursor RNA following cleavage at the poly(A) site, stopping when it reaches the Ur element, presumably attributable to a bound protein. Part of the function of this protein can be performed by the MS2 coat protein. Recruitment of coat protein to the ICR in the absence of the Ur element results in accumulation of an RNA equivalent to Ur-RNA, and restores trans-splicing. Only SL1, however, is used. Therefore, coat protein is sufficient for blocking the exonuclease and thereby allowing formation of a substrate for trans-splicing, but it lacks the ability to recruit the SL2 snRNP. Our results also demonstrate that MS2 coat protein can be used as an in vivo block to an exonuclease, which should have utility in mRNA stability studies.

Animals↗
PubMed2002

Multiple roles for SR proteins in trans splicing.

The trans-splicing reaction involves the association of 5' and 3' splice sites contained on separate transcripts. The mechanism by which these splice sites are juxtaposed during trans-spliceosome assembly and the role of SR proteins at each stage in this process have not been determined. Utilizing a system that allows for the separation of the RNA binding and RS domains of SR proteins, we have found that SR proteins are required for at least two stages of the trans-splicing reaction. They are important both prior to and subsequent to the addition of U2 snRNP to the 3' acceptor. In addition, we have demonstrated a role for RS domain phosphorylation in both of these activities. Dephosphorylation of the RS domain led to a block in U2 snRNP binding to the substrate. In a separate experiment, RS domain phosphorylation was also determined to be necessary for trans splicing to proceed on a substrate that had U2 snRNP already bound. This newly identified role for phosphorylated SR proteins post-U2-snRNP addition coincides with the recruitment of the 5' splice site contained on the SL RNP, suggesting a role for SR proteins in splice site communication in trans splicing.

Animals↗
PubMed2004

Topoisomerase I dissociates human immunodeficiency virus type 1 reverse transcriptase from genomic RNAs.

Both HIV-1 reverse transcriptase (RT) and topoisomerase I bind to structural RNAs and they cooperate to synthesize cDNA during the replication of HIV-1. In this study, we find that human topoisomerase I exclusively dissociated HIV-1 reverse transcriptase, which strongly binds to structural RNAs. Meanwhile, topoisomerase I did not dissociate either HIV-1 nucleocapsid proteins or murine leukemia virus RT which was bound to RNA. We propose that human topoisomerase I may regulate the binding of RT to RNAs and play a pivotal role in HIV-1 replication.

Base Sequence↗
PubMed2001

A complex containing CstF-64 and the SL2 snRNP connects mRNA 3' end formation and trans-splicing in C. elegans operons.

Polycistronic pre-mRNAs from Caenorhabditis elegans are processed by 3' end formation of the upstream mRNA and SL2-specific trans-splicing of the downstream mRNA. These processes usually occur within an approximately 100-nucleotide region and are mechanistically coupled. In this paper, we report a complex in C. elegans extracts containing the 3' end formation protein CstF-64 and the SL2 snRNP. This complex, immunoprecipitated with alphaCstF-64 antibody, contains SL2 RNA, but not SL1 RNA or other U snRNAs. Using mutational analysis we have been able to uncouple SL2 snRNP function and identity. SL2 RNA with a mutation in stem/loop III is functional in vivo as a trans-splice donor, but fails to splice to SL2-accepting trans-splice sites, suggesting that it has lost its identity as an SL2 snRNP. Importantly, stem/loop III mutations prevent association of SL2 RNA with CstF-64. In contrast, a mutation in stem II that inactivates the SL2 snRNP still permits complex formation with CstF-64. Therefore, SL2 RNA stem/loop III is required for both SL2 identity and formation of a complex containing CstF-64, but not for trans-splicing. These results provide a molecular framework for the coupling of 3' end formation and trans-splicing in the processing of polycistronic pre-mRNAs from C. elegans operons.

Animals↗
PubMed2006

U2 snRNA-protein contacts in purified human 17S U2 snRNPs and in spliceosomal A and B complexes.

The 17S U2 snRNP plays an essential role in branch point selection and catalysis during pre-mRNA splicing. Much remains to be learned about the molecular architecture of the U2 snRNP, including which proteins contact the functionally important 5' end of the U2 snRNA. Here, RNA-protein interactions within immunoaffinity-purified human 17S U2 snRNPs were analyzed by lead(II)-induced RNA cleavage and UV cross-linking. Contacts between the U2 snRNA and SF3a60, SF3b49, SF3b14a/p14 and SmG and SmB were detected. SF3b49 appears to make multiple contacts, interacting with the 5' end of U2 and nucleotides in loops I and IIb. SF3a60 also contacted different regions of the U2 snRNA, including the base of stem-loop I and a bulge in stem-loop III. Consistent with it contacting the pre-mRNA branch point adenosine, SF3b14a/p14 interacted with the U2 snRNA near the region that base pairs with the branch point sequence. A comparison of U2 cross-linking patterns obtained with 17S U2 snRNP versus purified spliceosomal A and B complexes revealed that RNA-protein interactions with stem-loop I and the branch site-interacting region of U2 are dynamic. These studies provide important insights into the molecular architecture of 17S U2 snRNPs and reveal U2 snRNP remodeling events during spliceosome assembly.

Cross-Linking Reagents↗
PubMed2007

Rapid test for the evaluation of the activity of the prodrug hydroxymethylnitrofurazone in the processing of Trypanosoma cruzi messenger RNAs.

No fully effective treatment has been developed since the discovery of Chagas' disease by Carlos Chagas in 1909. Since drug-resistant Trypanosoma cruzi strains are occurring and the current therapy is effectiveness in the acute phase but with various adverse side effects, more studies are needed to characterize the susceptibility of T. cruzi to new drugs. Many natural and/or synthetic substances showing trypanocidal activity have been used, even though they are not likely to be turned into clinically approved drugs. Originally, drug screening was performed using natural products, with only limited knowledge of the molecular mechanism involved in the development of diseases. Trans-splicing, which is unusual RNA processing reaction and occurs in nematodes and trypanosomes, implies the processing of polycistronic transcription units into individual mRNAs; a short transcript spliced leader (SL RNA) is trans-spliced to the acceptor pre-mRNA, giving origin to the mature mRNA. In the present study, permeable cells of T. cruzi epimastigote forms (Y, BOL and NCS strains) were treated to evaluate the interference of two drugs (hydroxymethylnitrofurazone - NFOH-121 and nitrofurazone) in the trans-splicing reaction using silver-stained PAGE analysis. Both drugs induced a significant reduction in RNA processing at concentrations from 5 to 12.5 microM. These data agreed with the biological findings, since the number of parasites decreased, especially with NFOH-121. This proposed methodology allows a rapid and cost-effective screening strategy for detecting drug interference in the trans-splicing mechanism of T. cruzi.

Animals↗
PubMed2006

Biochemical characterization of Trypanosoma brucei RNA polymerase II.

In Trypanosoma brucei, transcription by RNA polymerase II accounts for the expression of the spliced leader (SL) RNA and most protein coding mRNAs. To understand the regulation of RNA polymerase II transcription in these parasites, we have purified a transcriptionally active enzyme through affinity chromatography of its essential subunit, RPB4. The enzyme preparation is active in both promoter-independent and promoter-dependent in vitro transcription assays. Importantly, the enzyme is sensitive to alpha-amanitin inhibition, a hallmark of eukaryotic RNA polymerase II enzymes. Using mass spectrometric analysis we have identified the previously unobserved RPB12 subunit of T. brucei RNA polymerase II. TbRPB12 contains a conserved CX(2)CX(10-15)CX(2)C zinc binding motif that is characteristic of other eukaryotic RPB12 polypeptides. We also identified seven proteins that associate with T. brucei RNA polymerase II. While both bioinformatics and biochemical analysis have focused on the subunit structure of trypanosome RNA polymerases, this is the first study that reveals a functional RNA polymerase II enzyme.

Amanitins↗
PubMed1985

Effect of the tripartite leader on synthesis of a non-viral protein in an adenovirus 5 recombinant.

The EIa region of an Adenovirus 5 recombinant has been substituted by a modular gene encoding dihydrofolate reductase (DHFR). In this recombinant, the mouse DHFR cDNA was positioned behind sequences of the major late promoter and the complete tripartite leader. The leader sequences end in the normal 5' splice site (SS) of the third leader, so that RNA splicing joins the tripartite leader to a 3' splice site immediately upstream of the DHFR cDNA. At late stages of infection, high levels of DHFR mRNAs were synthesized. At early times in the late stage, this mRNA was efficiently translated; however, at later times translation of DHFR decreased probably due to poor competition with other late mRNAs. Synthesis of DHFR protein from an analogous Adenovirus 5 recombinant containing only the first late leader was studied in parallel. Equivalent levels of DHFR mRNA were expressed after infection with this recombinant virus; however, the efficiency of DHFR translation was at least 20 fold lower than that of the DHFR mRNA containing the tripartite leader. This suggests that the tripartite leader sequence is important for translation in the late stage of infection. As reported previously, the Ad5 recombinant containing only the first leader vastly overexpresses polypeptide IX from a novel mRNA, formed by the splicing of the first leader in the modular DHFR gene to the 3' splice site in the EIb region. Cells infected with this recombinant synthesize very little normal mRNA from the EIb region. Here, we demonstrated that coinfection of 293 cells with this recombinant and wild type Adenovirus 5 also results in decreased EIb mRNA synthesis. We propose that the overproduction of polypeptide IX suppresses mRNA expression from the EIb and IX promoter sites, probably by an autoregulation loop active during lytic growth.

Adenoviridae↗
PubMed2000

trans splicing of polycistronic Caenorhabditis elegans pre-mRNAs: analysis of the SL2 RNA.

Genes in Caenorhabditis elegans operons are transcribed as polycistronic pre-mRNAs in which downstream gene products are trans spliced to a specialized spliced leader, SL2. SL2 is donated by a 110-nucleotide RNA, SL2 RNA, present in the cell as an Sm-bound snRNP. SL2 RNA can be conceptually folded into a phylogenetically conserved three-stem-loop secondary structure. Here we report an in vivo mutational analysis of the SL2 RNA. Some sequences can be changed without consequence, while other changes result in a substantial loss of trans splicing. Interestingly, the spliced leader itself can be dramatically altered, such that the first stem-loop cannot form, with only a relatively small loss in trans-splicing efficiency. However, the primary sequence of stem II is crucial for SL2 trans splicing. Similarly, the conserved primary sequence of the third stem-loop plays a key role in trans splicing. While mutations in stem-loop III allow snRNP formation, a single nucleotide substitution in the loop prevents trans splicing. In contrast, the analogous region of SL1 RNA is not highly conserved, and its mutation does not abrogate function. Thus, stem-loop III appears to confer a specific function to SL2 RNA. Finally, an upstream sequence, previously predicted to be a proximal sequence element, is shown to be required for SL2 RNA expression.

Animals↗
PubMed2004

Failure to detect binding of Trypanosoma brucei SNAPc to U2 and U6 snRNA gene sequences by in vitro transcription competition and pull-down assays.

The small nuclear RNA (snRNA)-activating protein complex (SNAP(c)) is a multi-subunit transcription factor characterized in humans and Drosophila melanogaster. It binds to an upstream sequence element (USE) of snRNA gene promoters and activates both RNA polymerase (pol) II and III-mediated transcription of snRNA genes. The first identified and partially characterized transcription factor in a trypanosomatid organism appears to be a SNAP(c) homologue. It was identified in Leptomonas seymouri and shown to specifically interact with the USE of the RNA pol II-transcribed spliced leader (SL) RNA gene. Recently, chromatin immunoprecipitation and a gel shift assay suggested that L. seymouri SNAP(c) also interacts with RNA pol III-transcribed U2 and U6 snRNA genes. Previously, we have characterized and epitope-tagged the Trypanosoma brucei homologue of the SNAP50 subunit. Here, we show by in vitro transcription competition and promoter pull-down assays that TbSNAP50 binds to the SL RNA gene promoter and parasite-specifically to the ribosomal RNA gene promoter. Conversely, we did not detect binding of the factor to U2 and U6 snRNA gene sequences. Since U snRNA gene promoters are structurally conserved among trypanosomatids, our findings contrast those obtained in L. seymouri and suggest that trypanosomatid SNAP(c) is not involved in RNA pol III-mediated transcription of U snRNA genes.

Animals↗
PubMed2000

Identification of a novel RNA splicing pattern as a basis of restricted cell tropism of erythrovirus B19.

Prior studies on the transcription of erythrovirus B19 have identified a short leader sequence associated with all spliced viral transcripts. While some variability has been observed in the acceptor for this first intron, studies to date in both permissive and nonpermissive cell types have reported a unique splice donor site. In the semipermissive MB-02 cell line, we have found that splicing of this first intron proceeds almost exclusively via a cryptic CT donor downstream of the previously reported GT donor at nucleotide 406. The resulting messages for the viral structural proteins and 11-kDa protein are thereby made bicistronic, with the first expressible polypeptide being a 34 amino acid fusion of the NS-1 and 7.5-kDa proteins. The presence of an upstream open-reading frame on these messages is likely to block effective translation of the downstream structural protein products. We propose this as a significant mechanism in determining B19's tropism on the basis of host cell splicing machinery, and present evidence in support of this model. Additionally, this is the first report of usage of a noncanonical splice donor in B19, and to our knowledge the first report of a CT-AG splice in any system.

Alternative Splicing↗
PubMed1996

Evidence for the presence of a small U5-like RNA in active trans-spliceosomes of Trypanosoma brucei.

The existence of the Trypanosoma brucei 5' splice site on a small RNA of uniform sequence (the spliced leader or SL RNA) has allowed us to characterize the RNAs with which it interacts in vivo by psoralen crosslinking treatment. Analysis of the most abundant crosslinks formed by the SL RNA allowed us previously to identify the spliced leader-associated (SLA) RNA. The role of this RNA in trans-splicing, as well as the possible existence of an analogous RNA interaction in cis-splicing, is unknown. We show here that the 5' splice site region of the SL RNA is also crosslinked in vivo to a second small RNA. Although it is very small and lacks a 5' trimethylguanosine (TMG) cap, the SLA2RNA possesses counterparts of the conserved U5 snRNA stem-loop 1 and internal loop 1 sequence elements, as well as a potential trypanosome snRNA core protein binding site; these combined features meet the phylogenetic definition of U5 snRNA. Like U5, the SLA2 RNA forms an RNP complex with the U4 and U6 RNAs, and interacts with the 5' splice site region via its putative loop 1 sequence. In a final analogy with U5, the SLA2 RNA is found crosslinked to a molecule identical to the free 5' exon splicing intermediate. These data present a compelling case for the SLA2 RNA not only as an active trans-spliceosomal component, but also for its identification as the trypanosome U5 structural homolog. The presence of a U5-like RNA in this ancient eukaryote establishes the universality of the spliceosomal RNA core components.

Animals↗
PubMed2001

mRNA 5'-leader trans-splicing in the chordates.

We report the discovery of mRNA 5'-leader trans-splicing (SL trans-splicing) in the chordates. In the ascidian protochordate Ciona intestinalis, the mRNAs of at least seven genes undergo trans-splicing of a 16-nucleotide 5'-leader apparently derived from a 46-nucleotide RNA that shares features with previously characterized splice donor SL RNAs. SL trans-splicing was known previously to occur in several protist and metazoan phyla, however, this is the first report of SL trans-splicing within the deuterostome division of the metazoa. SL trans-splicing is not known to occur in the vertebrates. However, because ascidians are primitive chordates related to vertebrate ancestors, our findings raise the possibility of ancestral SL trans-splicing in the vertebrate lineage.

Animals↗
PubMed1984

Identification of a small RNA containing the trypanosome spliced leader: a donor of shared 5' sequences of trypanosomatid mRNAs?

The 35 nucleotide spliced leader (SL) sequence is found on the 5' end of numerous trypanosome mRNAs, yet the tandemly organized reiteration units encoding this leader are not detectably linked to any of these structural genes. Here we report the presence of a class of discrete small SL RNA molecules that are derived from the genomic SL reiteration units of Trypanosoma brucei, Trypanosoma cruzi, and Leptomonas collosoma. These small SL RNAs are 135, 105, and 95 nucleotides, respectively, and contain a 5'-terminal SL or SL-like sequence. S1 nuclease analyses demonstrate that these small SL RNAs are transcribed from continuous sequence within the respective SL reiteration units. With the exception of the SL sequence and a concensus donor splice site immediately following it, these small RNAs are not well conserved. We suggest that the small SL RNAs may function as a donor of the SL sequence in an intermolecular process that places the SL at the 5' terminus of many trypanosomatid mRNAs.

Animals↗
PubMed1995

Molecular analysis of Caenorhabditis elegans tcp-1, a gene encoding a chaperonin protein.

A Caenorhabditis elegans (Ce) homologue to the eukaryotic tcp-1 gene (encoding t-complex polypeptide-1) has been mapped, isolated and sequenced. Ce tcp-1 is a single-copy gene located on chromosome II. Nucleotide sequence analysis of the gene reveals the presence of four introns in the coding region and repetitive elements upstream from the start codon. The predicted Ce TCP-1 protein displays more than 60% amino-acid sequence identity to other eukaryotic TCP-1, suggesting a common origin and function for these proteins. The primary tcp-1 transcript undergoes trans-splicing to the spliced leader SL1 RNA, in addition to cis-splicing, to yield a single mRNA species of 1.9 kb. Northern blot analysis shows that unlike the evolutionarily related Hsp60 chaperonin genes, tcp-1 is not upregulated at elevated temperatures, but instead appears to be down-regulated. Additionally, the overall level of the tcp-1 transcript is approximately constant throughout the development of the nematode. The Ce chaperonin-containing TCP-1 (CCT) was identified. A protein extract made from Ce embryos was subjected to sucrose gradient fractionation and ATP-agarose chromatography. Western blot analysis of the purified protein fractions, using anti-mouse TCP-1 monoclonal antibody and antibodies raised against Ce TCP-1, reveals that Ce TCP-1 is a 57-kDa protein subunit of a high-molecular-mass complex capable of binding ATP.

Amino Acid Sequence↗
PubMed1987

The length but not the sequence of the polyoma virus late leader exon is important for both late RNA splicing and stability.

Polyoma virus late RNA processing provides a convenient model system in which to study the mechanics of splicing in vivo. In order to understand further the role of the untranslated "late leader" unit in late RNA processing we have constructed a group of polyoma viruses with deletions and substitutions in the leader exon. This has allowed us to determine that there is a minimum exon size required for both pre-mRNA splicing and stability in this system. We show here that the non-viability of a mutant (ALM) with a 9 base late leader unit is due to a general defect in late RNA splicing. In addition, ALM-infected cells show at least 40-fold depression in the accumulation of late nuclear RNA (spliced or unspliced). The ALM late promoter, however, functions nearly normally. Substituted leader variants with 51- to 96-base long exons of unrelated sequence are viable (G. Adami and G. Carmichael, J. Virol. 58, 417-425, 1986). We show here that late RNA from one of these substituted leader mutants (containing a 51-base leader exon) is spliced at wild type levels, with virtually no defect in accumulation. Thus, in the polyoma system, splice sites separated by only 9 bases can inhibit each others usage, presumably by steric interference. We suggest that this type of inhibition leads to extreme RNA instability.

Animals↗