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PubMed1984

A model for the excision of introns 1 and 2 from adenoviral major late pre-messenger RNAs.

Total steady-state RNA was extracted from nuclei of HeLa cells late after infection with adenovirus serotype 2. Most of the nuclear RNA is transcribed from the major late transcription unit (16.2 to 100.0 map units). To study the cleavage reactions involved in the splicing of leaders 1 and 2, we have used the S1 nuclease mapping technique with restriction fragments located in the region of intron 1 as DNA probes. The S1 mapping data showed that in total nuclear RNA, RNA species accumulate from which the 5' part of intron 1 has been excised, but which still contain the 3' part of the intron. This indicates that intron 1 can be removed in a stepwise fashion following the 5' to 3' direction. We have compared the nucleotide sequences from the ends of the putative processing intermediates. The internal cleavage sites do not resemble the consensus 5' or 3' splice site sequences. However, they show considerable homology to the sequence 5' A-T-G-A-T-G-G-C-A-T 3', which may act as a signal for internal cleavage. The intermediates are present in both the poly(A)+ and poly(A)- RNA fractions, although with different relative intensities. Primer extension experiments have been performed in which a primer, located with its left end in leader 2, is extended into intron 1. The results show that there may be a cleavage site as short as 35 nucleotides before the 3' splice site. Cleavage at the 3' splice site seems to be rapidly followed by ligation of leader 1 to leader 2. A model for RNA splicing based on these findings and data from the literature is presented.

Adenoviruses, Human↗
PubMed1997

Surprising diversity and distribution of spliced leader RNAs in flatworms.

Trans-splicing generates the mature 5' ends of certain mRNAs through the addition of a small spliced leader (SL) exon to pre-mRNAs. To search for novel flatworm spliced leaders, degenerate oligonucleotides and 5' RACE [corrected was used to isolate and characterize the 5' terminal sequences of enolase mRNAs in diverse flatworms. Several new spliced leaders and their SL RNA genes were identified, characterized, and compared. All parasitic trematodes examined trans-splice enolase. A primitive polyclad turbellarian, Stylochus zebra, also contains a trans-spliced enolase mRNA. The S. zebra SL is the longest SL yet identified, 51 nucleotides. Comparison of flatworm SLs indicates that they vary significantly in sequence and length. This suggests that neither spliced leader exon sequence nor size is likely to be essential for trans-splicing in flatworms. Flatworm SL RNAs have unusual Sm binding sites with characteristics distinct from other known flatworm snRNA Sm binding sites. Predicted flatworm SL RNA secondary structures show variation exhibiting 2-4 stem loops. Although limited in sequence similarity, phylogenetically conserved regions within the diverse flatworm SL RNAs suggest that they are likely to be derived from a common ancestor and provide information on potentially important SL RNA elements. The identification of a SL in a primitive flatworm suggests that trans-splicing may have been an ancestral feature in the phylum. Representative species of other early and more recent clades within the phylum, however, do not trans-splice enolase, nor do they or representatives of several other flatworm groups, have an SL RNA with a phylogenetically conserved region identified in the current study.

Amino Acid Sequence↗
PubMed1991

Insertion of part of an intron into the 5' untranslated region of a Caenorhabditis elegans gene converts it into a trans-spliced gene.

In nematodes, the RNA products of some genes are trans-spliced to a 22-nucleotide spliced leader (SL), while the RNA products of other genes are not. In Caenorhabditis elegans, there are two SLs, SL1 and SL2, donated by two distinct small nuclear ribonucleoprotein particles in a process functionally quite similar to nuclear intron removal. We demonstrate here that it is possible to convert a non-trans-spliced gene into a trans-spliced gene by placement of an intron missing only the 5' splice site into the 5' untranslated region. Stable transgenic strains were isolated expressing a gene in which 69 nucleotides of a vit-5 intron, including the 3' splice site, were inserted into the 5' untranslated region of a vit-2/vit-6 fusion gene. The RNA product of this gene was examined by primer extension and PCR amplification. Although the vit-2/vit-6 transgene product is not normally trans-spliced, the majority of transcripts from this altered gene were trans-spliced to SL1. We termed the region of a trans-spliced mRNA precursor between the 5' end and the first 3' splice site an "outron." Our results suggest that if a transcript begins with intronlike sequence followed by a 3' splice site, this alone may constitute an outron and be sufficient to demarcate a transcript as a trans-splice acceptor. These findings leave open the possibility that specific sequences are required to increase the efficiency of trans-splicing.

Animals↗
PubMed1978

Mapping the spliced and unspliced late lytic SV40 RNAs.

The sizes and map positions of the major late lytic SV40 cytoplasmic mRNAs and the abundant nuclear RNA species have been determined by the technique of Berk and Sharp (1977, 1978). From these experiments, the coding sequences (bodies) of the 16S and 19S late cytoplasmic SV40 RNAs have been located at 0.935-0.17 and 0.765-0.17 map units, respectively. The cytoplasmic 16S RNA molecules contain a leader sequence of approximately 210 nucleotides, corresponding to SV40 map positions 0.72-0.76 units, spliced to the coding sequences. In a population of the late 19S RNA molecules, there are several different leader segments, each spliced to the same coding sequences. The size of these 19S leader RNA segments was estimated to be 50-70, 100-120 and 200-210 nucleotides in length. The 5' ends of the 19S leader RNA segments were located at 0.72, 0.71, 0.695 and 0.69 map units. An analysis of the nuclear viral RNAs has provided insight into the biogenesis of the cytoplasmic messages. Poly (A)-containing nuclear RNA has a number of species in addition to those found in the cytoplasm. The 3' ends of the poly (A)-containing RNAs map at 0.17 SV40 units. The 5' ends of the more abundant nuclear molecules map approximately at 0.72, 0.70, 0.67, 0.64 and 0.59 units. Since these nuclear SV40 RNA molecules are both colinear with the viral DNA and larger than the cytoplasmic nRNAs, they may represent intermediates in a stepwise processing system. Alternatively, the variation in 5' ends of nuclear SV40 transcripts may represent a number of separate initiation sites for transcription. The presence of the intervening RNA sequences (between the leader and the coding sequences of the mature mRNAs) in these nuclear RNA molecules suggests that the synthesis of "spliced" SV40 RNA involves the direct transcription of the DNA sequences and the subsequent splicing out of the intervening seqment of RNA. Evaluation of the more abundant nonpolyadenylated nuclear RNA molecules showed that they have the same 5' ends as the poly (A)-containing nuclear RNAs. The 3' ends of the nonpolyadenylated RNA molecules map heterogeneously in a broad region extending beyond 0.28 map units. The presence of these long nuclear viral transcripts suggests that transcription of late SV40 RNA does not terminate at 0.17 map units. The location of poly (A) in mature cytoplasmic viral RNA at 0.17 map units suggest that poly (A) addition to RNA molecules may occur by a specific cleavage of the longer transcripts. Based on these analyses, we propose that the longer nonpolyadenylated viral RNA molecules in the nuclei of SV40-infected cells may represent the primary transcripts. While their 5' termini are being processed, the specific addition of poly (A) at 0.17 map units takes place. The polyadenylation of RNA is followed by splicing events to generate the cytoplasmic forms of SV40 mRNA.

Base Sequence↗
PubMed1986

A DNA insertion/deletion necessitates an aberrant RNA splice accounting for a mu heavy chain disease protein.

The human heavy chain disease protein BW is an immunoglobulin mu-chain variant whose amino terminus is initiated at the fifth amino acid of the first constant region domain. We cloned and analyzed both rearranged heavy chain alleles from BW leukemic cells to determine the molecular basis for this deleted protein. The phenotypically excluded heavy-chain allele possessed two intermediate recombinations of separate variable-diversity (V-D) and diversity-joining (D-J) junctions, neither of which were expressed. The productive allele, responsible for the mu chain, had a complete V-D-J4 recombination but as a result of a single-base deletion possessed stop codons within the variable region. More important, a small DNA insertion/deletion eliminated the J4 donor splice site. This necessitated an aberrant RNA splice between the leader region and the first constant region domain creating a shortened 2.35-kilobase muRNA. A recognition sequence for signal peptidase predicted a cleavage at the fifth amino acid of the first constant region domain. These molecular events are responsible for the truncated mu chain that lacks a variable region and fails to assemble light chains.

Alleles↗
PubMed1995

Expression of a TCR V beta 8.2 polypeptide from the unrearranged gene in a murine lymphoid precursor cell line.

The TCR in a mature T cell is a multimeric complex of TCR alpha and beta chains, and CD3 subunits. Functional TCR alpha and beta chains are encoded by genes that result from developmentally controlled somatic rearrangement events. By FACS analysis, we have detected a TCR V beta 8 protein on the surface of an immature lymphoid cell line, C1-V13D, that has all of its TCR genes in germline (unrearranged) configuration. RNA blot analysis detected a 1.4 kb polydenylated V beta 8 RNA in C1-V13D cells, but no expression of C beta was detected. Rapid amplification of 3' cDNA ends was used to clone an RNA that was initiated from the leader exon of the V beta 5.1 gene and spliced to the V exon of the V beta 8.2 gene. The putative sequence of the mature 10.8 kDa protein was entirely encoded by the V beta 8.2 exon. RT-PCR analysis confirmed that 97% of the V beta 8 RNA detected in C1-V13D cells was encoded by the V beta 8.2 gene, and only 3% by V beta 8.1 and V beta 8.3 genes. Furthermore, most of the V beta 8.2 RNA was spliced to the leader exon of the V beta 5.1 gene and not to the leader exon of the V beta 8.2 gene. The implications of preferential transcription from particular germline TCR genes for repertoire diversity and possible functions for proteins translated from germline TCR V beta genes are discussed.

Alternative Splicing↗
PubMed2000

Characterization of trans-splicing in Euglenoids.

We have looked for trans-splicing of nuclear mRNAs in several Euglenoid species. In Cyclidiopsis acus, Phacus curvicauda, Rhabdomonas costata and Menoidium pellucidum we showed that several premRNAs chosen at random are matured by a transsplicing process: we identified SL-RNA genes whose 5' ends (SLs for spliced leader-sequences) were transferred to the 5' extremities of mRNAs. The SL-RNA genes are located on repeated DNA fragments which also encode 5S rRNA in P. curvicauda and C. acus. The potential secondary structures of SL-RNAs are compared to those previously characterized in two other Euglenoids: Euglena gracilis and Entosiphon sulcatum. In another Euglenoid species, Distigma proteus, since none of the mRNAs examined were trans-spliced, it is possible that trans-splicing does not occur. Phylogeny based on 5S rRNA sequences suggests that the species which have, or have had, chloroplasts (E. gracilis, P. curvicauda, C. acus) diverged early from the others.

Animals↗
PubMed1985

Characterization of the Trypanosoma brucei 5S ribosomal RNA gene and transcript: the 5S rRNA is a spliced-leader-independent species.

Recent studies have shown that transcription occurs discontinuously for many genes in Trypanosoma brucei. To further investigate details of transcription in trypanosomes, the genes for the 5S ribosomal RNA from Trypanosoma brucei rhodesiense and Trypanosoma brucei brucei were cloned. Sequence analysis and Southern blotting showed the genes to be arranged in highly conserved tandem repeats of approx. 740 bp, which have no relation to the conserved 35-base spliced-leader repeat element. The genes contain internal control regions similar to 5S genes of other species, and studies of the 5S gene transcript show that it does not contain the conserved 35-base spliced-leader found at the 5' end of other trypanosome transcripts. Moreover, the 5S rRNA can be capped by guanylyltransferase from vaccinia virus, indicating that it has a 5' di- or triphosphate terminus. These results strongly suggest that the spliced-leader does not take part in the transcription of the 5S gene and that discontinuous transcription may be limited to particular classes of transcripts determined, as in other species, by the type of RNA polymerase used in their transcription. The DNA sequences of the 5S gene repeat from T.b. brucei and T.b. rhodesiense are presented, and their evolutionary significance is discussed.

Animals↗
PubMed1999

Identification of epi-1 locus as a laminin alpha chain gene in the nematode Caenorhabditis elegans and characterization of epi-1 mutant alleles.

A new genetic locus, epi-1, has been identified and mapped, which affects epithelialization of various tissues in the nematode, Caenorhabditis elegans. Seven independent epi-1 mutant alleles have been obtained. These mutants have a wide spectrum of abnormalities, all seem to be caused by a primary defect of basement membrane. We have identified the epi-1 gene as a structural gene of laminin alpha chain. The sequence analyses of the gene and cDNAs revealed that the gene consists of 15 exons and encodes a protein of 3704 amino acids in an open reading frame of 11115 base pairs. The nematode alpha chain is similar to its vertebrate and fly orthologs in the domain structure. The mRNA is trans-spliced to SL1 leader RNA as many of the nematode mRNAs. Mutation sites have been identified in four alleles. Two alleles have nonsense mutations and produce truncated proteins lacking the domain necessary for the formation of a heterotrimeric laminin molecule. The other two alleles have missense mutations in domains VI and IIIb, respectively.

Alleles↗
PubMed1993

Leptomonas seymouri as a model system for the analysis of gene expression in trypanosomatids.

Leptomonas seymouri, a monogenetic trypanosomatid originally isolated from Dysdercus suturellus (Hemiptera), was used to develop a reverse genetic system for trypanosomatid flagellates. In many eukaryotic cell types, reverse genetics has proven to be a powerful tool for defining structure/function relationships within genes. The mini-exon genes of trypanosomatids encode key components of all cellular mRNAs. This component is a 5' "leader" RNA that is spliced onto all mRNA precursors during mRNA formation within the cell nucleus. The data presented here indicate that structure/function relationships within the mini-exon gene can be probed using the molecular genetic system developed and characterized for L. seymouri.

Animals↗
PubMed2000

Unusual organization of the 5S ribosomal spacer in Dirofilaria repens: absence of a canonical spliced leader 1 sequence.

Comparing the 5S ribosomal RNA spacer of Dirofilaria repens and D. immitis, we found that in D. repens PCR generates two amplification products. In both products, the putative spliced leader 1 sequences (SL1) show some nucleotide substitutions compared to the canonical SL1 sequence of nematodes. SL I is a short sequence transferred to the 5' end of most messenger RNAs through a trans-splicing reaction. All the other filariae studied thus far amplify a single fragment showing a canonical SL1 sequence. D. repens appears thus peculiar.

Animals↗
PubMed1986

The 35-nucleotide spliced leader sequence is common to all trypanosome messenger RNA's.

In Trypanosomatidae the messenger RNA's (mRNA's) that code for the variant surface glycoproteins (VSG's), tubulins, calmodulin, and at least a subset of other proteins contain a common 35-nucleotide leader sequence at their 5' ends. Hybrid-arrested in vitro translation has been used to show that all mRNA's in both African and South American trypanosomes contain this 35-nucleotide sequence. Oligonucleotides complementary to this sequence blocked translation of all trypanosome mRNA's in a rabbit reticulocyte lysate system, but did not inhibit translation of mRNA's from other organisms lacking this sequence. An oligonucleotide complementary to the VSG mRNA downstream from the spliced leader sequence arrested only VSG synthesis. Thus, the 35-nucleotide leader sequence is a general feature of all trypanosome mRNA's. The high specificity of oligonucleotides complementary to the spliced leader for their target sequence suggests that analogues permeable to the cell membrane may be useful in the treatment of trypanosomal infections.

Base Sequence↗
PubMed2005

Messenger RNA processing sites in Trypanosoma brucei.

In Kinetoplastids, protein-coding genes are transcribed polycistronically by RNA polymerase II. Individual mature mRNAs are generated from polycistronic precursors by 5' trans splicing of a 39-nt capped leader RNA and 3' polyadenylation. It was previously known that trans splicing generally occurs at an AG dinucleotide downstream of a polypyrimidine tract, and that polyadenylation is coupled to downstream trans splicing. The few polyadenylation sites that had been examined were 100-400 nt upstream of the polypyrimidine tract which marked the adjacent trans splice site. We wished to define the sequence requirements for trypanosome mRNA processing more tightly and to generate a predictive algorithm. By scanning all available Trypanosoma brucei cDNAs for splicing and polyadenylation sites, we found that trans splicing generally occurs at the first AG following a polypyrimidine tract of 8-25 nt, giving rise to 5'-UTRs of a median length of 68 nt. We also found that in general, polyadenylation occurs at a position with one or more A residues located between 80 and 140 nt from the downstream polypyrimidine tract. These data were used to calibrate free parameters in a grammar model with distance constraints, enabling prediction of polyadenylation and trans splice sites for most protein-coding genes in the trypanosome genome. The data from the genome analysis and the program are available from: .

5' Untranslated Regions↗
PubMed1984

Apolipoprotein II messenger RNA. Transcriptional and splicing heterogeneity yields six 5'-untranslated leader sequences.

The structure of the mRNA for apolipoprotein II (apo-II), a major avian estrogen-responsive yolk protein, has been investigated. Primer extension using a cDNA probe-primer revealed six forms of mature apo-II mRNA. S1 and primer extension analysis with intron probes showed that only three of these forms arise due to multiple sites of transcription initiation on the apo-II gene. To investigate the basis for the additional forms of apo-II mRNA, we examined their nucleotide sequence. The sequence obtained between -10 and -42, relative to translation initiation, is identical to that reported by Wieringa et al. (Wieringa, B., AB, G., and Gruber, M. (1981) Nucleic Acids Res. 9, 469-499). At position -43, however, sequence heterogeneity appears. The minor form of the sequence starting at -43 and extending in a 5' direction corresponds exactly to the published mRNA sequence. The major form of the sequence has the insertion, 5'-CAG-3', at this position. The apparent basis for this heterogeneity is an unusual intron-exon border which violates the consensus sequence found in comparable positions in most eukaryotic genes by the existence of two adjacent 5'-CAG-3' triplets after the pyrimidine (Y)-rich track. The processing ratio between intron removal at the upstream and downstream AG dinucleotide is approximately 2.5:1. This result demonstrates that the splicing mechanism allows spatial flexibility in the positioning of the highly conserved YAG triplet within the consensus sequence at the 3' splice site.

Animals↗
PubMed1990

Stable expression of the bacterial neor gene in Leishmania enriettii.

Molecular genetic studies in parasitic protozoa have been hindered by the lack of methods for the introduction and expression of modified or foreign genes in these organisms. Two recent reports described the transient expression of the bacterial chloramphenicol acetyl transferase (CAT) gene under the control of parasite-specific sequences. We now describe the stable expression of a selectable marker, the gene for neomycin resistance (neor) in Leishmania enriettii. A chimaeric gene containing the neor gene inserted between two alpha-tubulin intergenic sequences was introduced into the cells and drug-resistant L. enriettii were observed which stably expressed the neor gene. One goal of this work was to analyse the sequences necessary for trans-splicing of messenger RNA, as trypanosomatids have a novel process of RNA trans-splicing, described initially in Trypanosome brucei and subsequently in several other trypanosomatids, including L. enriettii. Many trypanosomatid genes are arranged in tandem arrays and the intergenic sequences contain both the splice acceptor site for the addition of the spliced leader sequence and a putative polyadenylation site. Messenger RNA isolated from several different neor L. enrietti lines contained the spliced leader sequence joined to the neor gene at the position of the splice acceptor site in the alpha-tubulin intergenic sequence. The neor mRNA was also polyadenylated. Plasmid DNA is present within the drug-resistant organisms and appears to be extrachromosomal. The development of these methods allows the functional analysis of sequences necessary for trans-splicing.

Animals↗
PubMed1997

Heterogeneous nuclear ribonucleoprotein A1 binds to the transcription-regulatory region of mouse hepatitis virus RNA.

A cellular protein, previously described as p35/38, binds to the complementary (-)-strand of the leader RNA and intergenic (IG) sequence of mouse hepatitis virus (MHV) RNA. The extent of the binding of this protein to IG sites correlates with the efficiency of the subgenomic mRNA transcription from that IG site, suggesting that it is a requisite transcription factor. We have purified this protein and determined by partial peptide sequencing that it is heterogeneous nuclear ribonucleoprotein (hnRNP) A1, an abundant, primarily nuclear protein. hnRNP A1 shuttles between the nucleus and cytoplasm and plays a role in the regulation of alternative RNA splicing. The MHV(-)-strand leader and IG sequences conform to the consensus binding motifs of hnRNP A1. Recombinant hnRNP A1 bound to these two RNA regions in vitro in a sequence-specific manner. During MHV infection, hnRNP A1 relocalizes from the nucleus to the cytoplasm, where viral replication occurs. These data suggest that hnRNP A1 is a cellular factor that regulates the RNA-dependent RNA transcription of the virus.

Animals↗
PubMed1991

RNA-protein complexes mediate in vitro capping of the spliced-leader primary transcript and U-RNAs in Trypanosoma cruzi.

A 39-nucleotide spliced leader (SL) is joined to the 5' ends of trypanosome mRNAs in a bimolecular or trans-splicing process. The SL in Trypanosoma cruzi is transcribed as an approximately 110-nucleotide RNA (SL-RNA or SL primary transcript) bearing the 39-nucleotide SL at the 5' end. The SL-RNA is 5' capped by a guanylyltransferase activity prior to trans-splicing and trypanosome mRNAs thus obtain their mature caps from the SL by trans-splicing. We have previously characterized a guanylyltransferase activity from T. cruzi nuclear extracts and shown that this capping activity has an unusual ATP dependence and an apparent specificity for the SL-RNA and U-RNAs. Herein, we show that the capping activity sediments as a 12-15S particle during velocity sedimentation in glycerol gradients and fractionates as a greater than 150-kDa particle during large-pore gel filtration chromatography. Moreover, the endogenous substrate RNAs--the SL-RNA and U-RNAs--consistently copurify with the capping activity, suggesting that the activity and the substrates form a ribonucleoprotein particle. The capping activity and substrate RNAs are not dissociated in isopycnic Cs2SO4 gradients and band at a density expected for an RNA-protein complex, confirming the existence of ribonucleoprotein particles bearing both the activity and its substrate RNAs. Finally, we partially purified these ribonucleoprotein particles and showed that the capping activity remains ATP dependent and highly specific for the SL-RNA and the U-RNAs. These observations are consistent with the hypothesis that one of the functions of trans-splicing is for mRNA capping.

Animals↗