Biomedical subjects

P A Maroney

Publications and source records attributed to P A Maroney.

At least 19 recordsLinked to original sources

PubMed2001

3' splice site recognition in nematode trans-splicing involves enhancer-dependent recruitment of U2 snRNP.

Trans-splicing requires that 5' and 3' splice sites be independently recognized. Here, we have used mutational analyses and a sensitive nuclease protection assay to determine the mechanism of trans-3' splice site recognition in vitro. Efficient recognition of the 3' splice site is dependent upon both the sequence of the 3' splice site itself and enhancer elements located in the 3' exon. We show that the presence of three distinct classes of enhancers results in increased binding of U2 snRNP to the branchpoint region. Several lines of evidence strongly suggest that the increased binding of U2 snRNP is mediated by U2AF. These results expand the roles of enhancers in constitutive splicing and provide direct support for the recruitment model of enhancer function.

Animals↗

PubMed2000

Functional recognition of 5' splice site by U4/U6.U5 tri-snRNP defines a novel ATP-dependent step in early spliceosome assembly.

A sensitive assay based on competition between cis-and trans-splicing suggested that factors in addition to U1 snRNP were important for early 5' splice site recognition. Cross-linking and physical protection experiments revealed a functionally important interaction between U4/U6.U5 tri-snRNP and the 5' splice site, which unexpectedly was not dependent upon prior binding of U2 snRNP to the branch point. The early 5' splice site/tri-snRNP interaction requires ATP, occurs in both nematode and HeLa cell extracts, and involves sequence-specific interactions between the highly conserved splicing factor Prp8 and the 5' splice site. We propose that U1 and U5 snRNPs functionally collaborate to recognize and define the 5' splice site prior to establishment of communication with the 3' splice site.

Adenosine Triphosphate↗

PubMed2000

Nuclease protection of RNAs containing site-specific labels: a rapid method for mapping RNA-protein interactions.

This report describes a method that combines nuclease protection and site-specific labeling to determine sites and extents of RNA-protein interactions. The utility of the method is demonstrated by the analysis of the binding of factors to the 3' splice site region of a pre-mRNA labeled at three specific positions. This "reverse footprinting" technique should be widely applicable to a variety of questions concerning RNA-protein interactions.

Binding Sites↗

PubMed1996

Direct analysis of nematode cis- and trans-spliceosomes: a functional role for U5 snRNA in spliced leader addition trans-splicing and the identification of novel Sm snRNPs.

Most nuclear pre-mRNAs in nematodes are processed by both cis- and trans-splicing. In trans-splicing, the 5' terminal exon, the spliced leader sequence (SL), is derived from a trans-splicing specific Sm snRNP, the SL RNP. Because U snRNPs are required cofactors for trans-splicing, and because this processing reaction proceeds via a two-step reaction pathway identical to that of cis-splicing, it has long been assumed that trans-splicing is catalyzed in a complex analogous to the cis-spliceosome. However, similarities or differences between cis- and trans-spliceosomes have not been established. In particular, the role of U5 snRNP in trans-splicing has been unclear. Here, we have used affinity selection to analyze the U snRNA constituents of nematode cis- and trans-spliceosomes. We find that U5 snRNP is an integral component of the trans-spliceosome and, using site-specific crosslinking, we show that U5 snRNP establishes specific Interactions with the SL RNA exon. We also identify two novel Sm snRNPs that are enriched in both cis- and trans-spliceosomes. Finally, we provide evidence that a SL RNP-containing multi-snRNP (SL, U4, U5, and U6 RNPs) may be a functional precursor in trans-spliceosome assembly.

Animals↗

PubMed1996

Multiple requirements for nematode spliced leader RNP function in trans-splicing.

The 5' exon donor in nematode trans-splicing, the SL RNA, is a small (approximately 100 nt) RNA that resembles cis-spliceosomal U snRNAs. Extensive analyses of the RNA sequence requirements for SL RNA function have revealed four essential elements, the core Sm binding site, three nucleotides immediately downstream of this site, a region of Stem-loop II, and a 5' splice site. Although these elements are necessary and sufficient for SL RNA function in vitro, their respective roles in promoting SL RNA activity have not been elucidated. Furthermore, although it has been shown that assembly of the SL RNA into an Sm RNP is a prerequisite for function, the protein composition of the SL RNP has not been determined. Here, we have used oligoribonucleotide affinity to purify the SL RNP and find that it contains core Sm proteins as well as four specific proteins (175, 40, 30, and 28 kDa). Using in vitro assembly assays; we show that association of the 175- and 30-kDa SL-specific proteins correlates with SL RNP function in trans-splicing. Binding of these proteins depends upon the sequence of the core Sm binding site; SL RNAs containing the U1 snRNA Sm binding site assemble into Sm RNPs that contain core, but not SL-specific proteins. Furthermore, mutational and thiophosphate interference approaches reveal that both the primary nucleotide sequence and a specific phosphate oxygen within a segment of Stemloop II of the SL RNA are required for function. Finally, mutational activation of an unusual cryptic 5' splice site within the SL sequence itself suggests that U5 snRNA may play a primary role in selecting and specifying the 5' splice site in SL addition trans-splicing.

Animals↗

PubMed1995

U6 snRNA function in nuclear pre-mRNA splicing: a phosphorothioate interference analysis of the U6 phosphate backbone.

U6 snRNA is essential for and may participate in the catalysis of pre-mRNA splicing. Extensive mutational analyses in several systems have identified nucleotides essential for U6 function in splicing; however, relatively little is known regarding the role of the U6 phosphate backbone. We previously described a mutation in a nematode U6 snRNA that causes it to be used as a splicing substrate within the spliceosome. This unusual reaction has made it possible to apply modification interference analysis to U6 function. Here, we have used phosphorothioate substitution to identify pro-R oxygens throughout the U6 backbone that are necessary for the first and/or second catalytic steps of splicing. Four pro-R oxygens are important for the first step; of these only two appear to be required. One additional pro-R oxygen is uniquely required for the second step. The two pro-R oxygens critical for the first step of splicing are in the helix 1b U2/U6 interaction region and the intramolecular stem-loop of U6, respectively. A comparison of the positions of these two pro-R oxygens with those found to be critical for autocatalytic excision of a group II intron suggests a possible functional similarity between U6 snRNA and domain V of group II introns.

Animals↗

PubMed1995

Most mRNAs in the nematode Ascaris lumbricoides are trans-spliced: a role for spliced leader addition in translational efficiency.

Some pre-mRNAs in nematodes are processed by trans-splicing. In this reaction, a 22-nt 5' terminal exon (the spliced leader, SL) and its associated 2,2,7-trimethylguanosine cap are acquired from a specialized Sm snRNP, the SL RNP. Although it has been evident for many years that not all nematode mRNAs contain the SL sequence, the prevalence of trans-spliced mRNAs has, with the exception of Caenorhabditis elegans, not been determined. To address this question in an organism amenable to biochemical analysis, we have prepared a message-dependent protein synthesis system from developing embryos of the parasitic nematode, Ascaris lumbricoides. Using this system, we have used both hybrid-arrest and hybrid-selection approaches to show that the vast majority (80-90%) of A. lumbricoides mRNAs contain the SL sequence and therefore are processed by trans-splicing. Furthermore, to examine the effect of SL addition on translation, we have measured levels of protein synthesis in extracts programmed with a variety of synthetic mRNAs. We find that the SL sequence itself and its associated hypermethylated cap functionally collaborate to enhance translational efficiency, presumably at the level of initiation of protein synthesis. These results indicate that trans-splicing plays a larger role in nematode gene expression than previously suspected.

Animals↗

PubMed1994

Transcription of a nematode U1 small nuclear RNA in vitro. 3'-end formation requires cis-acting elements within the coding sequence.

We have used block-substitution mutagenesis and in vitro transcription assays to identify cis-acting DNA sequence elements important for initiation and 3'-end formation of a U1 small nuclear RNA (snRNA) in the parasitic nematode Ascaris lumbricoides. Efficient initiation of synthesis by RNA polymerase II requires a compact element centered approximately 50 base pairs upstream of the transcriptional start site. Surprisingly, 3'-end formation of U1 snRNAs synthesized in vitro is solely dependent upon elements within the U1 coding sequence. In all other systems studied thus far, 3'-end formation of U snRNAs requires signals present in the 3'-flanking region. We also show that sequence elements that direct 3'-end formation of the A. lumbricoides trans-spliced leader RNA function when RNA synthesis is initiated from the U1 promoter. These results indicate that 3'-end formation of U snRNAs in nematodes is mechanistically distinct from the analogous process in higher eukaryotes.

Animals↗

PubMed1993

Functional reconstitution of U6 snRNA in nematode cis- and trans-splicing: U6 can serve as both a branch acceptor and a 5' exon.

Maturation of nuclear pre-mRNAs in nematodes requires both cis- and trans-splicing. Both processing pathways involve analogous two-step phosphotransfer reactions and both are dependent upon the integrity of U6 snRNA. We have developed a functional reconstitution assay to assess the U6 snRNA sequence requirements for cis- and trans-splicing. Branch formation between the splicing substrates and U6 snRNA was observed. The frequency of this event was greatly enhanced when a highly conserved sequence in U6 snRNA was altered by mutation. In cis- and trans-splicing reactions reconstituted with this mutant U6 snRNA the liberated exon of U6 proceeded through the second step of splicing using the appropriate splice acceptor sites. These results demonstrate covalent interactions between a U snRNA required for splicing and a splicing substrate, and they provide evidence for an unexpected degree of catalytic flexibility within the spliceosome.

Animals↗

PubMed1992

Interaction of U6 snRNA with a sequence required for function of the nematode SL RNA in trans-splicing.

Nematode trans-spliced leader (SL) RNAs are composed of two domains, an exon [the 22-nucleotide spliced leader] and a small nuclear RNA (snRNA)-like sequence. Participation in vitro of the spliced leader RNA in trans-splicing reactions is independent of the exon sequence or size and instead depends on features contained in the snRNA-like domain of the molecule. Chemical modification interference analysis has revealed that two short sequence elements in the snRNA-like domain are necessary for SL RNA activity. These elements are sufficient for such activity because when added to a 72-nucleotide fragment of a nematode U1 snRNA, this hybrid RNA could participate in trans-splicing reactions in vitro. One of the critical sequence elements may function by base-pairing with U6 snRNA, an essential U snRNA for both cis- and trans-splicing.

Animals↗

PubMed1991

Multiple cis-acting elements are required for RNA polymerase III transcription of the gene encoding H1 RNA, the RNA component of human RNase P.

In humans, the H1 RNA, the RNA subunit of RNase P, is synthesized by RNA polymerase III. We have used block replacement mutagenesis to identify the sequences necessary for in vitro transcription of H1 RNA. We find that multiple cis-acting elements located in the H1 RNA 5'-flanking region are necessary for H1 RNA synthesis; no internal sequences are essential. Required cis-acting elements include sequences resembling proximal sequence element, distal sequence element, and TATA motifs. In this respect, the H1 RNA promoter is similar in structure to the promoters of the genes encoding the U6 snRNA, the 7 SK RNA and the MRP RNA. However, our mutational analysis indicates that the H1 promoter is unexpectedly complex, with several additional cis-acting elements spanning nearly 70 base pairs of the H1 RNA gene 5'-flanking sequence.

Base Sequence↗

PubMed1991

U small nuclear ribonucleoprotein requirements for nematode cis- and trans-splicing in vitro.

In nematodes, a fraction of mRNAs acquires a common 22-nucleotide 5'-terminal spliced leader sequence via a trans-splicing reaction. The same premessenger RNAs which receive the spliced leader are also processed by conventional cis-splicing. Whole cell extracts prepared from synchronous embryos of the parasitic nematode Ascaris lumbricoides catalyze both cis- and trans-splicing. We have used this cell-free system and oligodeoxynucleotide directed RNase H digestion to assess the U small nuclear RNA requirements for nematode cis- and trans-splicing. These experiments indicated that both cis- and trans-splicing require intact U2 and U4/U6 small nuclear ribonucleoproteins (snRNPs). However, whereas cis-splicing displays the expected requirement for an intact U1 snRNP, trans-splicing is unaffected when approximately 90% of U1 snRNP is degraded. These results suggest that 5' splice site identification differs in nematode cis- and trans-splicing.

Animals↗

PubMed1991

Intramolecular base pairing between the nematode spliced leader and its 5' splice site is not essential for trans-splicing in vitro.

The spliced leader RNAs of both trypanosomes and nematodes can form similar secondary structures where the trans-splice donor site is involved in intramolecular base pairing with the spliced leader sequence. It has been proposed that this base pairing could serve to activate autonomously the SL RNA splice donor site. Here, we have examined exon requirements for trans-splicing in a nematode cell free system. Complete disruption of secondary structure interactions at and around the trans-splice donor site did not affect the ability of the SL RNA to function in trans-splicing. In addition, the highly conserved 22 nt sequence could be productively replaced by artificial exons ranging in size from 2 to 246 nucleotides. These results reinforce the view that the 'intron' portion of the SL RNA functions as an independent Sm snRNP whose role is to deliver exon sequences to the trans-spliceosome.

Animals↗

PubMed1990

Protective efficacy of a cloned Brugia malayi antigen in a mouse model of microfilaremia.

Immunization of mice with irradiated Brugia larvae or parasite extracts has been shown to induce partial resistance to microfilaremia and enhance clearance of infective larvae. We recently reported the cloning of a 548 amino acid 62-kDa Brugia malayi Ag identified on the basis of reactivity with antisera to a subset of protective microfilarial Ag. Our study describes the protective efficacy against microfilaremia in mice, immunogenicity, and parasite stage-specificity of this candidate vaccine molecule. Immunization of Swiss or BALB/c mice with 1 to 3 micrograms of a 92-kDa trpE fusion protein encoding amino acids 1-479 reduced the intensity of microfilaremia by 40 to 60% compared to control animals given buffer or bacterial trpE (p less than 0.01 to 0.001). Mice immunized with the 92-kDa fusion protein developed delayed-type hypersensitivity reactivity to B. malayi as assessed by enhanced footpad swelling 24 and 48 h after intradermal injection of adult worm extract and in vitro lymph node mononuclear cell proliferation (3H-thymidine uptake) in response to the fusion protein (mean +/- SD stimulation index 4.7 +/- 0.8 vs 2.0 +/- 1.4 for trpE, p less than 0.05). Proliferative responses of lymph node cells coincubated with three other fusion proteins corresponding to the filarial protein truncated from its carboxyl-terminus suggest that dominant T cell epitopes of the 62-kDa Ag are encompassed by amino acids 437-479. Rabbit antibody to the 92-kDa trpE fusion protein immunoprecipitated a 62-kDa polypeptide from [35S] methionine biosynthetically labeled B. malayi microfilariae, adult female, and adult male worms. These data indicate that a recombinant Ag expressed in several developmental stages of B. malayi is capable of inducing partial resistance against microfilariae and Ag-specific T cell responses in mice.

Animals↗

PubMed1990

Trans splicing of nematode pre-messenger RNA in vitro.

In nematodes, a fraction of mRNAs contains a common 22 nucleotide 5' terminal spliced leader (SL) sequence derived by trans splicing. Here, we show that a cell-free extract prepared from developing embryos of the parasitic nematode Ascaris lumbricoides catalyzes accurate and efficient SL addition to a synthetic pre-mRNA at an authentic trans splice acceptor site. SL addition occurs via a trans splicing reaction that proceeds through Y-branched intermediates. The branchpoint is located at either of two adenosine residues located 18 and 19 nucleotides upstream of the splice acceptor site.

Animals↗

PubMed1990

The nematode spliced leader RNA participates in trans-splicing as an Sm snRNP.

The trans-spliced leader RNA (SL RNA) of nematodes resembles U snRNAs both in cap structure and in the presence of a consensus Sm binding site. We show here that synthetic SL RNA, synthesized by in vitro transcription, is efficiently used as a spliced leader donor in trans-splicing reactions catalyzed by a cell free extract prepared from developing embryos of the parasitic nematode, Ascaris lumbricoides. Efficient utilization of synthetic SL RNA requires a functional Sm binding site. Mutations within the Sm binding sequence that prevent immunoprecipitation by Sm antisera and prevent cap trimethylation abolish trans-splicing. The effect on trans-splicing is not due to undermethylation of the cap structure.

Animals↗

PubMed1990

Transcription of a nematode trans-spliced leader RNA requires internal elements for both initiation and 3' end-formation.

We have used block substitution mutagenesis and in vitro transcription to define sequence elements important for efficient initiation and 3' end-formation of the trans-spliced leader RNA (SL RNA) of the parasitic nematode Ascaris lumbricoides. These experiments indicate that the SL RNA has an unusual promoter structure containing elements which include the 22 nt trans-spliced leader exon itself. Efficient transcription is correlated with the binding of a factor to the 22 nt (SL) sequence; mutations within the SL which abolish transcription lead to a loss in binding of this factor. In addition to internal sequences, synthesis of SL RNA in vitro requires an element centered 50 bases upstream of the cap site. Mutations within this element dramatically affect the level of SL RNA synthesis but do not affect accuracy of initiation. Finally, all of the information required for accurate 3' end-formation of SL RNA lies within the transcribed region. Thus, the arrangement of sequences necessary for the synthesis of SL RNAs does not resemble that of sequences important for the synthesis of vertebrate U snRNAs despite the similarities between SL RNAs and U snRNAs.

Animals↗

PubMed1990

Transcription and cap trimethylation of a nematode spliced leader RNA in a cell-free system.

Maturation of a fraction of mRNAs in nematodes involves the acquisition of a common 5' terminal spliced leader sequence derived from a nonpolyadenylylated spliced leader RNA by trans splicing. We have developed a cell-free system prepared from Ascaris lumbricoides embryos that accurately and efficiently synthesized the spliced leader RNA of A. lumbricoides. Transcription of the spliced leader RNA was catalyzed by RNA polymerase II, and the majority of the spliced leader RNAs synthesized in vitro possessed a trimethylguanosine cap structure identical to that found on in vivo-synthesized spliced leader RNA.

Animals↗