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PubMed1979

Rapid sequence determination of late simian virus 40 16S mRNA leader by using inhibitors of reverse transcriptase.

A method for the determination of the primary structure of spliced mRNA junction and leader sequences is described. By analogy to the DNA sequencing procedure of Sanger et al. [Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Natl. Acad. Sci USA 74, 5463--5467], we use 2",3'-dideoxynucleoside triphosphates as chain-terminating inhibitors of the reverse transcriptase (RNA-dependent DNA polymerase) reaction. By using specific DNA restriction fragments as primers in combination with this technique, we have determined the sequence of the spliced junction between the body and the leader sequence of the 16S late mRNA of simian virus 40. The method described should be of general utility in mapping spliced mRNA regions for which the corresponding protein sequence (if any) is unknown.

Base Sequence↗
PubMed1982

The orderly splicing of the first three leaders of the adenovirus-2 major late transcript.

A strategy based on the hybridization of labeled nucleoplasmic RNA to a short cloned cDNA probe was devised to study the ligation of the three first leader sequences (Le1, Le2, Le3) of the major late adenovirus-2 transcript. The hybridized RNA was subsequently fractionated by electrophoresis and identified with the aid of restriction fragments of the DNA probe. The ligations were shown to occur stepwise and in an orderly fashion. Le1 and Le2 were first ligated without detectable lag time. The tripartite leader was formed after a lag time of 10-15 min probably due, for a large part, to the stepwise excision of the intervening sequence between Le2 and Le3. The possible processing intermediate Le2-Le3 was not detected.

Adenoviruses, Human↗
PubMed1979

Synthesis and processing of adenoviral RNA in isolated nuclei.

Adenoviral RNA sequences synthesized in nuclei isolated during the late phase of productive infection comprise, besides virus-associated, VA, RNA, five major species, ranging in size from approximately 13S to 55S. The latter RNA species is of the length predicted if the major transcriptional unit expressed during the late phase were completely copied in vitro. Some 30% of the RNA sequences labeled in vitro are polyadenylated, and about one-third of the polyadenylated RNA is virus specific. Hybridization analysis of the sequences immediately adjacent to polyadenylic acid in late RNA labeled in isolated nuclei suggests that polyadenylation in vitro occurs at the same sites recognized within the cell. The polyadenylic acid-containing viral RNA sequences made in isolated nuclei are found in three major species of RNA, sedimenting at approximately 28S, 18S, and 13S. These sizes are remarkably similar to those reported for late mRNA species, suggesting that additional processing steps can occur in isolated nuclei. Hybridization of RNA to XhoI fragments of adenovirus type 2 DNA transferred to nitrocellulose filters reveals that sequences complementary to the region from 22.0 to 26.5 units present in 55S RNA are absent from all smaller species, suggesting that the smaller RNA species labeled in isolated nuclei are generated by splicing. The splicing events necessary to generate the 5' leader segment common to the majority of late adenoviral mRNA species are shown to be performed correctly in isolated nuclei.

Adenoviruses, Human↗
PubMed1988

A 22-nucleotide spliced leader sequence in the human parasitic nematode Brugia malayi is identical to the trans-spliced leader exon in Caenorhabditis elegans.

The mRNAs encoding a 63-kDa antigen in the human parasitic nematode Brugia Malayi contain a spliced leader sequence of 22 nucleotides (nt) that is identical to the trans-spliced leader found on certain actin mRNAs in the distantly related nematode Caenorhabditis elegans. The 22-nt sequence does not appear to be encoded near the 63-kDa genes but is present in multiple copies in several locations within the parasite genome, including the 5S rRNA gene repeat. The 5S-linked copies of the 22-nt sequence are transcribed to yield a 109-nt nonpolyadenylated RNA with the 22-nt leader sequence at its 5' end. We suggest that the 22-nt leader is acquired by 63-kDa antigen mRNAs through trans-splicing. These results indicate that trans-splicing is widespread in nematodes and argue for the functional significance of the 22-nt spliced leader exon in nematode mRNA metabolism.

Animals↗
PubMed1991

Splice site skipping in polyomavirus late pre-mRNA processing.

Polyomavirus late nuclear primary transcripts contain tandem repeats of the late strand of the viral genome, as a result of inefficient transcription termination and polyadenylation. Pre-mRNA processing involves the splicing of short noncoding late leader exons to each other (removing genome-length introns) and the splicing of the last leader to a coding body exon (such as for the major virion structural protein, VP1). As a result, cytoplasmic mRNAs contain 1 to 12 tandem leader exons at their 5' ends that are followed by a single coding exon. To understand more about how polyomavirus exons are spliced together, we studied a double-genome construct consisting of two tandem but nonidentical polyomavirus late transcription units. The alternating leader exons are distinguishable from one another but retain identical flanking RNA-processing signals, as for the alternating VP1 exons. We transfected this construct and derivatives of it into mouse cells and determined which leader exons are spliced to which others and which VP1 exons are utilized. Results showed that leader exons are almost never skipped during splicing and are spliced sequentially to one another. On the other hand, VP1 exons were often skipped, with the VP1 exon closest to the polyadenylation site splicing to the nearest upstream leader exon. Splice site replacement experiments showed that VP1 exon skipping is not due to a relative weakness of its 3' splice site or to any sequence upstream of the VP1 3' splice site. Exon skipping is also not the result of sequences within the VP1 exon. Rather, VP1 3' splice site skipping can be eliminated by replacing the inefficient late polyadenylation signal with an efficient one, or by inserting a 5' splice site between the VP1 3' splice site and the late polyadenylation site. Thus, sequences that compose the distal border of the VP1 exon can influence usage of the upstream 3' splice site.

3T3 Cells↗
PubMed1997

PCR-based amplification of total cDNA with high fidelity and high yield from minute amounts of parasite RNA.

cDNA, synthesised from total RNA from Acanthocheilonema viteae, was amplified by PCR with a primer derived from the spliced leader 1 sequence of nematodes and oligo-dT. Due to the great number of side products observed in the reaction, a biotinylated oligo-dT primer was used for cDNA-synthesis and the first cycles of PCR. After binding of the PCR products to streptavidin/paramagnetic particles, the (+)strands of the cDNAs were recovered and reamplified. Analysis of the PCR products obtained revealed the presence of full-length cDNAs of at least 1.7 kbp in size in amplified total cDNA from microfilariae, postinfective L3, and adult worms. The total cDNA, from only 20 ex vivo recovered postinfective L3, was efficiently amplified.

Animals↗
PubMed1989

Heat-shock disruption of trans-splicing in trypanosomes: effect on Hsp70, Hsp85 and tubulin mRNA synthesis.

The synthesis of most, if not all, mRNA in trypanosomes involves the splicing in trans of two RNA molecules. One of these includes the common 5' mini-exon or spliced-leader sequence, while the other contains the complete protein-coding exon sequence for a given gene. We have recently shown that trans-splicing of tubulin transcripts is disrupted by heat shock, whereas trans-splicing of Hsp70 mRNA continues unabated, despite the increased expression of this latter gene [Muhich and Boothroyd, Mol. Cell Biol. 8 (1988) 3837-3846]. In this report we show that, in addition to being polyadenylated, the dimeric beta-alpha tubulin transcripts which accumulate as a result of heat shock have a correctly processed 5'-end (through mini-exon addition), and that the extent of disruption is proportional to the degree of heat shock. We further show that mRNA levels for two evolutionarily-conserved heat-shock proteins, Hsp70 and Hsp85, are increased under the splicing-disruptive conditions of heat shock.

Animals↗
PubMed1995

Alternative RNA splicing generates diversity of neuropeptide expression in the brain of the snail Lymnaea: in situ analysis of mutually exclusive transcripts of the FMRFamide gene.

In the CNS of the snail Lymnaea stagnalis, Phe-Met-Arg-Phe-amide (FMRFamide)-like and additional novel neuropeptides are encoded by a common, multi-exon gene. This complex locus, comprising at least five exons, is subject to post-transcriptional regulation at the level of alternative RNA splicing. Our aim was first to analyse the pattern by which exons of this neuropeptide locus combine during splicing of the primary RNA transcript, and second to investigate the functional significance of splicing by mapping the expression and neuronal localization in the CNS of the alternative mRNA transcripts, in the context of defined neuronal networks and single identified neurons. The approach was a combination of comparative in situ hybridization and immunocytochemistry, using a battery of exon-specific oligonucleotides and anti-peptide antisera. The analysis illustrated that exons III, IV and V were always coexpressed and colocalized whereas the expression of exon II was always differential and mutually exclusive. Both sets of exons were, however, coexpressed with exon I: the total number of exon I-expressing neurons was equal to the combined number of neurons expressing exon III/IV/V and neurons expressing exon II. In addition, it was revealed that the extreme 5' of exon II, encoding a potential hydrophobic leader signal, was not expressed in the CNS of Lymnaea but was apparently spliced out during RNA processing. Both mRNA transcripts of the FMRFamide locus, type 1 (exons I/II) and type 2 (exons I/III/IV/V), were translated in the CNS and the resulting protein precursors were also expressed in a mutually exclusive fashion, as were their respective transcripts. The expression of alternative transcripts within identified networks or neuronal clusters was heterogeneous, as exemplified by the cardiorespiratory network. On the basis of this work and a previous cDNA analysis, we put forward a revised model of differential splicing and expression of the FMRFamide gene in the CNS of Lymnaea.

Amino Acid Sequence↗
PubMed1988

Multiple genomic defects result in an alternative RNA splice creating a human gamma H chain disease protein.

Heavy chain diseases (HCD) are human lymphoproliferative disorders in which a clonal B cell population produces Ig molecules made of truncated H chains without associated L chain. We characterized the rearranged H chain gene and its mRNA from the leukemic cells of a patient (RIV) with gamma-HCD. The abnormal RIV serum Ig consisted of shortened, dimeric gamma 1-chains which had an amino terminus within the hinge region. RIV lymphoblasts possessed a foreshortened (1200 bp) gamma 1-mRNA which had sequences for only the leader, hinge, second, and third constant region domains (CH2 + CH3), but lacked variable (VH) and CH1 information. Sequence of the productive gamma 1 allele revealed it had undergone VH-JH and H chain class switch recombinations. However, normal RNA splice sites had been eliminated by a DNA insertion/deletion (VH acceptor site), mutations (JH donor site), or a large deletion (CH1 region). Inserted sequences were of non-Ig and apparently non-genomic origin. These DNA alterations resulted in aberrant mRNA processing in which the leader region was spliced directly to the hinge region, accounting for the HCD protein.

Chromosome Aberrations↗
PubMed1984

Transcription strategy of coronaviruses: fusion of non-contiguous sequences during mRNA synthesis.

MHV replicates in the cell cytoplasm and viral genetic information is expressed in infected cells as one genomic sized RNA ( mRNA1 ) and six subgenomic mRNAs. The seven RNAs were assumed to have common 3' ends of the size of RNA7 , the smallest RNA. The data reported here, show that this model is too simple and that the mRNAs are composed of a leader and body sequence. Electron microscopic analysis of hybrids formed between single stranded cDNA copied from mRNA7 and genomic RNA or mRNA6 shows that genomic RNA, mRNA6 and mRNA7 have common 5' terminal sequences. Furthermore, nucleotide sequence analysis shows that the nucleotide sequence of the 5' end of mRNA7 diverges from the corresponding region of the genome just upstream from the initiation codon of the nucleocapsid gene. Because the synthesis of each mRNA is inactivated by UV irradiation in proportion to its own length, the subgenomic mRNAs are apparently not produced by the processing of larger RNAs. The available data have to be explained by translocation of the polymerase/leader complex to specific internal positions on the negative strand. In this way the leader and body sequences are joined together by a mechanism completely different from conventional RNA splicing but nevertheless giving the same end result.

Animals↗
PubMed1997

Transcription of germline VH gene elements by normal human fetal liver.

Transcription of the gene elements that form the variable region of immunoglobulin heavy chains has been proposed to represent the process that controls access for the recombination enzymes in their sequential steps of catalysis. Evidence for germline transcription of VH gene elements, as part of VH to DJH recombination, has been limited to transcripts of only a few gene elements. We have examined normal fetal liver mRNA by Northern blotting and present evidence for germline transcripts from six human VH gene families. The candidate VH4 transcripts have been confirmed as germline transcripts by hybridization with 3' flanking sequences that would have been removed by recombination from mature VHDJH genes. The candidate transcripts for VH1, VH3, VH4 and VH6 have been confirmed by polymerase chain reaction amplification with primers from the 3' flanking sequences of these gene families and determination of the sequence of these products. Determination of sequence from two clones of VH1, VH3 and VH4 indicates that more than one gene from each of these families is transcribed. PCR amplification of VH4 and VH6 with primers specific for the leader sequence (exon 1) and 3' flanking sequence indicate that these transcripts are spliced, representing RNA processing. Germline transcripts from these families are also present in normal human bone marrow. These results indicate that transcriptional activation of germline VH gene elements is a general phenomenon in tissues undergoing V to DJ recombination.

Adult↗
PubMed1980

Definition of a novel promoter for the major adenovirus-associated virus mRNA.

A 660 nucleotide adenovirus-associated virus type 2 (AAV2) DNA fragment which encodes the 5' terminal leader and the entire intervening sequence of the major viral mRNA has been cloned into pBR322, and its primary sequence has been determined. The 5' terminal viral mRNA sequence was deduced by sequencing the reverse-transcriptase cDNA extension product of a 5' end-labeled DNA primer complementary to the RNA 5' terminal region. From combined DNA and RNA sequence analyses (which confirm our previous mapping data) we conclude that the major AAV2 transcript contains a 5' terminal leader sequence about 55 nucleotides in length encoded from a continuous region of DNA (near position 39 on the viral genome) 320 bases from the RNA body. The DNA sequences of the splice junctions are similar to those found for other class II genes. No other nucleotide sequence, indicative of promotion at another (upstream) site, is present at the 5' terminus. The DNA region encoding and flanking the leader sequence displays structural features expected for a class II gene promoter, including the canonical ATATAA sequence 23-25 bases upstream from the presumed initiation site. When the cloned viral DNA fragment is transcribed in vitro by RNA polymerase II in a cell-free system, a transcript is produced with a 5' end that is similar or identical to that found on the in vivo mRNA. Taken together these data strongly suggest that the major polysomal RNA may be generated from a transcription unit with a promoter at position 39, even though this transcription unit is part of a larger transcription unit with an upstream promoter near position 6. This indication of overlapping transcription units with independent promoters provides a major new insight into parvovirus gene expression.

Base Sequence↗
PubMed1988

Structure and transcription of the actin gene of Trypanosoma brucei.

In Trypanosoma brucei, the actin gene is present in a cluster of two, three, or four tandemly linked copies, depending on the strain. Each cluster seems to exist in two allelic versions, as suggested by the polymorphism of both gene number and restriction fragment length in the DNA from cloned trypanosomes. The amplification of the gene copy number probably occurs through unequal sister chromatid exchange. The chromosomes harboring the actin genes belong to the large size class. The coding sequence was 1,128 nucleotides long and showed 60 to 70% homology to other eucaryotic actin genes. Surprisingly, this homology seemed weaker with Trypanosoma congolense, Trypanosoma cruzi, Trypanosoma vivax, Trypanosoma mega, or Leishmania actin-specific sequences. The mRNA was around 1.6 kilobases long and was synthesized at the same level in bloodstream and procyclic forms of the parasite. Large RNA precursors, up to 7.7 kilobases, were found in a pattern identical in strains containing either two or three gene copies. Probing of the flanking regions of the gene with either steady-state or in vitro transcripts, as well as S1 nuclease protection and primer extension experiments, allowed mapping of the 3' splice site of the actin mRNA, 38 nucleotides upstream from the translation initiation codon. A variably sized poly(dT) tract was found about 30 base pairs ahead of the splice site. The largest detected actin mRNA precursor seemed to give rise to at least two additional stable mRNAs. The RNA polymerase transcribing the actin gene exhibited the same sensitivity to inhibition by alpha-amanitin as that transcribing both the spliced leader and the bulk of polyadenylated mRNAs.

Actins↗
PubMed1992

Bodo caudatus medRNA and 5S rRNA genes: tandem arrangement and phylogenetic analyses.

The Bodo caudatus mini-exon-derived RNA gene repeat has been isolated following PCR amplification. The DNA sequence of the mini-exon fits the trypanosomatid mini-exon consensus, supporting inclusion of Bodo in this group. The B. caudatus mini-exon repeat also contains the 5S ribosomal RNA gene, an organization found in the trypanosome T. rangeli and five genera of nematodes. Phylogenetic analysis of both mini-exon-derived RNA gene and 5S gene sequences show that the free-living B. caudatus is more closely related to the monogenetic Crithidia than the digenetic Trypanosoma. Similarity between the Euglena gracilis trans-spliced leader and trypanosomatid mini-exon sequences was also noted during these comparisons.

Animals↗
PubMed1979

Comparison of late mRNA splicing among class B and class C adenoviruses.

Adenovirus class B (Ad3 and Ad7) and class C (Ad1, Ad5, and Ad6) late r-strand mRNA's were found to have segmented 5' leaders. These leaders were very similar among serotype within a class but differed in sequence from the leaders on late mRNA's of a different class. However, the leader components of class B viruses mapped at essentially the same map coordinates as those of class C viruses. The 5' coordinates of the main bodies of class B messages to which the tripartite leaders are attached as well as the map positions of several of their early mRNA's were very similar to those of Ad2 transcripts. Infrequent examples of late r-strand polysomal RNAs of Ad3 and Ad7 had, in addition to the three common leader segments, a fourth leader segment derived from RNA encoded at various sites between the second and third leaders. The extra components formed several distinct groups. These molecules are presumably intermediates in the splicing processes that generate mature messages.

Adenoviruses, Human↗
PubMed1995

cis-splicing and polyadenylation of actin RNA can precede 5' trans-splicing in nematodes.

In nematodes many precursor RNAs undergo both cis-splicing (intron removal) and trans-splicing (spliced leader addition) via molecular mechanisms thought to be very similar. We examined the temporal order of trans- and cis-splicing by analyzing cDNAs of partially processed transcripts from an actin gene of the parasitic nematode, Onchocerca volvulus, which has five short introns. We found that in this model gene system trans-splicing can follow cis-splicing and polyadenylation, and we obtained no evidence for alternative pathways of splicing. This result is in contrast to the tubulin genes of African trypanosomes where trans-splicing precedes polyadenylation (1).

Actins↗
PubMed2005

Expression of estrogen receptor ESR1 and its 46-kDa variant in the gubernaculum testis.

Testicular descent corresponds to migration of the testis from the abdominal cavity to the scrotum and is essential for proper functioning of the testis. Recent advances in the characterization of estrogen receptor (ESR) subtypes and isoforms in various tissues prompted us to study ESRs within the gubernaculum testis, a structure involved in testicular descent. In the rat gubernaculum, we searched for ESR alpha (Esr1) and beta (Esr2) and for the androgen receptor (Ar), androgens being known to regulate testicular descent. Reverse transcription-polymerase chain reaction (RT-PCR) revealed that Esr1, Esr2, and Ar mRNAs were all expressed in the gubernaculum. Using PEETA (Primer extension, Electrophoresis, Elution, Tailing, and Amplification), we established that all Esr1 leader exons, previously identified in other organs, such as the uterus and pituitary, were transcribed in the gubernaculum, with the major form being O/B. The RNA protection assays, RT-PCR, and Western blot experiments revealed that isoform-specific mRNA transcripts generated by alternative splicing of the C-leader sequence on coding exons 1 and 2 of the Esr1 gene gave the 46- and 66-kDa ESR1 proteins. The ESR1 and AR proteins were found to colocalize in the parenchymal cells of the gubernaculum early in development, whereas AR also was strongly expressed in the muscular cells, both during fetal and postnatal life. The ESR2 protein was weakly expressed, principally in the muscular cells, but only once testicular descent had occurred. The levels of the 46-kDa ESR1 variant (ER46) exceeded those of the 66-kDa ESR1 form (ER66) at periods when the gubernaculum developed. Conversely, the 66-kDa form appears to predominate clearly when the gubernaculum growth was low or completed. The possible role of estrogens on the modulation of the androgen-dependent growth of the gubernaculum and, more widely, on testicular descent is discussed.

Alternative Splicing↗