Biomedical subjects

T Blumenthal

Publications and source records attributed to T Blumenthal.

At least 19 recordsLinked to original sources

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↗

PubMed2001

Interplay between AAUAAA and the trans-splice site in processing of a Caenorhabditis elegans operon pre-mRNA.

About half of Caenorhabditis elegans genes have a 1-2 bp mismatch to the canonical AAUAAA hexamer that signals 3' end formation. One rare variant, AGUAAA, is found at the 3' end of the mai-1 gene, the first gene in an operon also containing gpd-2 and gpd-3. When we expressed this operon under heat shock control, 3' end formation dependent on the AGUAAA was very inefficient, but could be rescued by a single bp change to create a perfect AAUAAA. When AGUAAA was present, most 3' ends formed at a different site, 100 bp farther downstream, right at the gpd-2 trans-splice site. Surprisingly, 3' end formation at this site did not require any observable match to the AAUAAA consensus. It is possible that 3' end formation at this site occurs by a novel mechanism--trans-splicing-dependent cleavage--as deletion of the trans-splice site prevented 3' end formation here. Changing the AGUAAA to AAUAAA also influenced the trans-splicing process: with AGUAAA, most of the gpd-2 product was trans-spliced to SL1, rather than SL2, which is normally used at downstream operon trans-splice sites. However, with AAUAAA, SL2 trans-splicing of gpd-2 was increased. Our results imply that (1) the AAUAAA consensus controls 3' end formation frequency in C. elegans; (2) the AAUAAA is important in determining SL2 trans-splicing events more than 100 bp downstream; and (3) in some circumstances, 3' end formation may occur by a trans-splicing-dependent mechanism.

Animals↗

PubMed2001

Intercistronic region required for polycistronic pre-mRNA processing in Caenorhabditis elegans.

In Caenorhabditis elegans, polycistronic pre-mRNAs are processed by cleavage and polyadenylation at the 3' ends of the upstream genes and trans splicing, generally to the specialized spliced leader SL2, at the 5' ends of the downstream genes. Previous studies have indicated a relationship between these two events in the processing of a heat shock-induced gpd-2-gpd-3 polycistronic pre-mRNA. Here, we report mutational analysis of the intercistronic region of this operon by linker scan analysis. Surprisingly, no sequences downstream of the 3' end were important for 3'-end formation. In contrast, a U-rich (Ur) element located 29 bp downstream of the site of 3'-end formation was shown to be important for downstream mRNA biosynthesis. This approximately 20-bp element is sufficient for SL2 trans splicing and mRNA accumulation when transplanted to a heterologous context. Furthermore, when the downstream gene was replaced by a gene from another organism, no loss of trans-splicing specificity was observed, suggesting that the Ur element may be the primary signal required for downstream mRNA processing.

Animals↗

PubMed2000

Functional characterization of five eIF4E isoforms in Caenorhabditis elegans.

Recognition of the 5'-cap structure of mRNA by eIF4E is a critical step in the recruitment of most mRNAs to the ribosome. In Caenorhabditis elegans, approximately 70% of mRNAs contain an unusual 2,2,7-trimethylguanosine cap structure as a result of trans-splicing onto the 5' end of the pre-mRNA. The characterization of three eIF4E isoforms in C. elegans (IFE-1, IFE-2, and IFE-3) was reported previously. The present study describes two more eIF4E isoforms expressed in C. elegans, IFE-4 and IFE-5. We analyzed the requirement of each isoform for viability by RNA interference. IFE-3, the most closely related to mammalian eIF4E-1, binds only 7-methylguanosine caps and is essential for viability. In contrast, three closely related isoforms (IFE-1, IFE-2, and IFE-5) bind 2,2, 7-trimethylguanosine caps and are partially redundant, but at least one functional isoform is required for viability. IFE-4, which binds only 7-methylguanosine caps, is most closely related to an unusual eIF4E isoform found in plants (nCBP) and mammals (4E-HP) and is not essential for viability in any combination of IFE knockout. ife-2, ife-3, ife-4, and ife-5 mRNAs are themselves trans-spliced to SL1 spliced leaders. ife-1 mRNA is trans-spliced to an SL2 leader, indicating that its gene resides in a downstream position of an operon.

Amino Acid Sequence↗

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↗

PubMed1999

Both subunits of U2AF recognize the 3' splice site in Caenorhabditis elegans.

Introns are defined by sequences that bind components of the splicing machinery. The branchpoint consensus, polypyrimidine (poly(Y)) tract, and AG at the splice boundary comprise the mammalian 3' splice site. Although the AG is crucial for the recognition of introns with relatively short poly(Y) tracts, which are termed 'AG-dependent introns', the molecule responsible for AG recognition has never been identified. A key player in 3' splice site definition is the essential heterodimeric splicing factor U2AF, which facilitates the interaction of the U2 small nuclear ribonucleoprotein particle with the branch point. The U2AF subunit with a relative molecular mass (Mr 65K) of 65,000 (U2AF65) binds to the poly(Y) tract, whereas the role of the 35K subunit (U2AF35) has not been clearly defined. It is not required for splicing in vitro but it plays a critical role in vivo. Caenorhabditis elegans introns have a highly conserved U4CAG/ R at their 3' splice sites instead of branch-point and poly(Y) consensus sequences. Nevertheless, C. elegans has U2AF, 12). Here we show that both U2AF subunits crosslink to the 3' splice site. Our results suggest that the U2AF65-U2AF35 complex identifies the U4CAG/R, with U2AF35 being responsible for recognition of the canonical AG.

Animals↗

PubMed1999

An exon that prevents transport of a mature mRNA.

In Caenorhabditis elegans, pre-mRNA for the essential splicing factor U2AF65 sometimes is spliced to produce an RNA that includes an extra 216-bp internal exon, exon 3. Inclusion of exon 3 inserts an in-frame stop codon, yet this RNA is not subject to SMG-mediated RNA surveillance. To test whether exon 3 causes RNA to remain nuclear and thereby escape decay, we inserted it into the 3' untranslated region of a gfp reporter gene. Although exon 3 did not affect accumulation or processing of the mRNA, it dramatically suppressed expression of green fluorescent protein (GFP). We showed by in situ hybridization that exon 3-containing gfp RNA is retained in the nucleus. Intriguingly, exon 3 contains 10 matches to the 8-bp 3' splice-site consensus. We hypothesized that U2AF might recognize this octamer and thereby prevent export. This idea is supported by RNA interference experiments in which reduced levels of U2AF resulted in a small burst of gfp expression.

Animals↗

PubMed1999

Numerical fourier transform spectroscopy of EMG half-waves: fragmentary-decomposition-based approach to nonstationary signal analysis.

A nonstationary signal analysis technique is introduced, which regards an oscillatory physiological signal as a sum of its fragments, presented in the form of a fragmentary decomposition (FD). The virtue of FD is that it is free of the necessity to choose a priori the basis functions intended for signal analysis or synthesis. FD uses an unchanged signal fragment between adjacent zero-crossings, as a natural basis function called the half-wave function (HWF). To show that such a function is a physically meaningful object, Fourier transform methods were employed, supported by the similar basis function (SBF) algorithm, which provides the means for numerical Fourier transform spectroscopy of separate half-waves and their frequency domain description in terms of both amplitude and phase. The application of this method to parameter identification of 751 EMG half-waves from the eye blink EMG records of ten normal subjects showed that HWF's frequency domain image represents a Gaussian distribution, which applies over a defined range of relative frequencies. This empirical evidence shows that HWFs are produced by a specific system of first-order nonlinear differential equations, whose dependency on a number of random factors is characteristic of deterministic chaos. The particular form of solutions indicates that statistical regularities relevant to the central limit theorem are likely to underlie the genesis of the mass potentials studied. FD shows potential utility in a range of nonstationary physiological signals.

Adolescent↗

PubMed1999

U2AF35 is encoded by an essential gene clustered in an operon with RRM/cyclophilin in Caenorhabditis elegans.

In most species the 3' splice site is recognized initially by an interaction between the two-subunit splicing factor U2AF with the polypyrimidine (poly(Y)) tract that results in recruitment of the U2 snRNP to the branch-point consensus just upstream. In contrast, in Caenorhabditis elegans, both the poly(Y) tract and the branch-point consensus sequences are missing, apparently replaced by the highly conserved U4CAG/R 3' splice site consensus. Nevertheless C. elegans U2AF65 is very similar to its mammalian and fly counterparts and may recognize the 3' splice site consensus. Here we report the cloning of the C. elegans U2AF35 gene, uaf-2. We show that it lacks an identifiable RS domain, which, in flies, has been shown to play a role in RNA binding, but it contains an extended glycine-rich stretch at its C-terminus. uaf-2 is in an operon with cyp-13, a gene that encodes a cyclophilin with an RRM domain at its N-terminus. We demonstrate by RNA interference that both U2AF genes, uaf-1 (which encodes U2AF65) and uaf-2, are required for viability, whereas cyp-13 is apparently not.

Amino Acid Sequence↗

PubMed1999

SL1 trans splicing and 3'-end formation in a novel class of Caenorhabditis elegans operon.

Many Caenorhabditis elegans genes exist in operons in which polycistronic precursors are processed by cleavage at the 3' ends of upstream genes and trans splicing 100 to 400 nucleotides away, at the 5' ends of downstream genes, to generate monocistronic messages. Of the two spliced leaders, SL1 is trans spliced to the 5' ends of upstream genes, whereas SL2 is reserved for downstream genes in operons. However, there are isolated examples of what appears to be a different sort of operon, in which trans splicing is exclusively to SL1 and there is no intercistronic region; the polyadenylation signal is only a few base pairs upstream of the trans-splice site. We have analyzed the processing of an operon of this type by inserting the central part of mes-6/cks-1 into an SL2-type operon. In this novel context, cks-1 is trans spliced only to SL1, and mes-6 3'-end formation occurs normally, demonstrating that this unique mode of processing is indeed intrinsic to this kind of operon, which we herein designate "SL1-type." An exceptionally long polypyrimidine tract found in the 3' untranslated regions of the three known SL1-type operons is shown to be required for the accumulation of both upstream and downstream mRNAs. Mutations of the trans-splice and poly(A) signals indicate that the two processes are independent and in competition, presumably due to their close proximity, raising the possibility that production of upstream and downstream mRNAs is mutually exclusive.

Animals↗

PubMed1998

Gene clusters and polycistronic transcription in eukaryotes.

Sometimes genes are arranged nonrandomly on the chromosomes of eukaryotes. This review considers instances of gene clusters in which two genes or more are expressed from a single promoter. This includes cases in which a polycistronic pre-mRNA is processed to make monocistronic mRNAs in nematodes, as well as isolated examples of polycistronic mRNAs found in mammals, flies, and perhaps plants.

Alternative Splicing↗

PubMed1997

Operons and SL2 trans-splicing exist in nematodes outside the genus Caenorhabditis.

The genomes of most eukaryotes are composed of genes arranged on the chromosomes without regard to function, with each gene transcribed from a promoter at its 5' end. However, the genome of the free-living nematode Caenorhabditis elegans contains numerous polycistronic clusters similar to bacterial operons in which the genes are transcribed sequentially from a single promoter at the 5' end of the cluster. The resulting polycistronic pre-mRNAs are processed into monocistronic mRNAs by conventional 3' end formation, cleavage, and polyadenylation, accompanied by trans-splicing with a specialized spliced leader (SL), SL2. To determine whether this mode of gene organization and expression, apparently unique among the animals, occurs in other species, we have investigated genes in a distantly related free-living rhabditid nematode in the genus Dolichorhabditis (strain CEW1). We have identified both SL1 and SL2 RNAs in this species. In addition, we have sequenced a Dolichorhabditis genomic region containing a gene cluster with all of the characteristics of the C. elegans operons. We show that the downstream gene is trans-spliced to SL2. We also present evidence that suggests that these two genes are also clustered in the C. elegans and Caenorhabditis briggsae genomes. Thus, it appears that the arrangement of genes in operons pre-dates the divergence of the genus Caenorhabditis from the other genera in the family Rhabditidae, and may be more widespread than is currently appreciated.

Animals↗

PubMed1997

ELT-1, a GATA-like transcription factor, is required for epidermal cell fates in Caenorhabditis elegans embryos.

Epidermal cells are generated during Caenorhabditis elegans embryogenesis by several distinct lineage patterns. These patterns are controlled by maternal genes that determine the identities of early embryonic blastomeres. We show that the embryonically expressed gene elt-1, which was shown previously to encode a GATA-like transcription factor, is required for the production of epidermal cells by each of these lineages. Depending on their lineage history, cells that become epidermal in wild-type embryos become either neurons or muscle cells in elt-1 mutant embryos. The ELT-1 protein is expressed in epidermal cells and in their precursors. We propose that elt-1 functions at an early step in the specification of epidermal cell fates.

Amino Acid Sequence↗

PubMed1997

Cloning of Caenorhabditis U2AF65: an alternatively spliced RNA containing a novel exon.

The U2 small nuclear ribonucleoprotein particle (snRNP) auxiliary factor, U2AF, is an essential splicing factor required for recognition of the polypyrimidine tract and subsequent U2 snRNP assembly at the branch point. Because Caenorhabditis elegans introns lack both polypyrimidine tract and branch point consensus sequences but have a very highly conserved UUUUCAG/R consensus at their 3' splice sites, we hypothesized that U2AF might serve to recognize this sequence and thus promote intron recognition in C. elegans. Here we report the cloning of the gene for the large subunit of U2AF, uaf-1. Three classes of cDNA were identified. In the most abundant class the open reading frame is similar to that for the U2AF65 from mammals and flies. The remaining two classes result from an alternative splicing event in which an exon containing an in-frame stop codon is inserted near the beginning of the second RNA recognition motif. However, this alternative mRNA is apparently not translated. Interestingly, the inserted exon contains 10 matches to the 3' splice site consensus. To determine whether this feature is conserved, we sequenced uaf-1 from the related nematode Caenorhabditis briggsae. It is composed of six exons, including an alternatively spliced third exon interrupting the gene at the same location as in C. elegans. uaf-1 is contained in an operon with the rab-18 gene in both species. Although the alternative exons from the two species are not highly conserved and would not encode related polypeptides, the C. briggsae alternative exon has 18 matches to the 3' splice site consensus. We hypothesize that the array of 3' splice site-like sequences in the pre-mRNA and alternatively spliced exon may have a regulatory role. The alternatively spliced RNA accumulates at high levels following starvation, suggesting that this RNA may represent an adaption for reducing U2AF65 levels when pre-mRNA levels are low.

Alternative Splicing↗

PubMed1997

Relationship between 3' end formation and SL2-specific trans-splicing in polycistronic Caenorhabditis elegans pre-mRNA processing.

About 25% of the genes in the nematode Caenorhabditis elegans are in operons, polycistronic transcription units in which the genes are only 100-400 bp apart. The operon pre-mRNAs are processed into monocistronic mRNAs by a combination of cleavage and polyadenylation at the 3' end of the upstream mRNA and SL2 trans-splicing at the 5' end of the downstream mRNA. To determine whether 3' end formation and SL2 trans-splicing are coupled mechanistically, we tested a gpd-2/gpd-3 operon construct driven by a C. elegans heat shock promoter, and measured the effects of inhibition of 3' end formation and/or trans-splicing on the processing of the polycistronic RNA in vivo. The results indicate that proper 3' end formation of the upstream mRNA in an operon is required for SL2-specificity of downstream mRNA trans-splicing. In contrast, trans-splicing of the downstream mRNA is not necessary for correct 3' end formation of the upstream mRNA. In addition, shortening the distance between the 5' cap and the AAUAAA of gpd-2 (the upstream gene) decreases the efficiency of 3' end formation and is accompanied by a replacement of SL2 with SL1 at the trans-splice site of gpd-3, the downstream gene. These results indicate that SL2 trans-splicing, in C. elegans, is coupled mechanistically to 3' end formation in the processing of polycistronic pre-mRNAs.

Animals↗

PubMed1996

Gene structure and organization in Caenorhabditis elegans.

The sequencing of the 100 Mb Caenorhabditis elegans genome-containing approximately 14,000 genes-is approximately 50% complete. One of its most interesting features is its compactness; introns and intergenic distances are unusually small and, surprisingly, approximately 25% of genes are contained in polycistronic transcription units (operons) with only approximately 100 bp between genes.

Animals↗

PubMed1996

Comparison of a vitellogenin gene between two distantly related rhabditid nematode species.

Three vitellogenin genes from the free-living nematode Caenorhabditis elegans have previously been characterized at the molecular level. In order to study evolutionary relationships within this poorly understood taxon, we have cloned a vitellogenin gene, CEW1-vit-6, from a distantly related species belonging to the same family as C. elegans. Screening of a genomic library with a probe to total poly(A+) RNA yielded three clones that hybridized more intensely than all others, and all three corresponded to a single gene homologous to C. elegans vit-6. Comparison of CEW1-vit-6 with Ce-vit-6 reveals both strong similarities and surprising differences. Life Ce-vit-6, the gene is about 5 kb long and contains four unusually small introns (38-41 nt), but only one interrupts the gene at the same location as a Ce-vit-6 intron. The promoter region contains five matches to Vitellogenin Promoter Element 1 (VPE1) and no matches to VPE2, both previously shown to be required for vit gene transcription in C. elegans. Codon usage is in general similar to that of the Ce-vit genes, but a few codon biases are quite different. Alignment of the CEW1-vit-6 protein with Ce-vit-6 and Ce-vit-2 products suggests the existence of two domains which have evolved at different rates. Sequence comparison shows that nematode vitellogenins are much more closely related to vertebrate than to insect vitellogenins.

Amino Acid Sequence↗

PubMed1996

Functional analysis of an intron 3' splice site in Caenorhabditis elegans.

Caenorhabditis elegans introns typically lack both branch point and polypyrimidine tract consensus sequences utilized in other organisms for intron recognition. However, most introns have an unusually long, highly conserved consensus, UUUCAG/R, at the 3' splice site. This site can be recognized even when the -1G is changed to A. To determine how the 3' splice site is defined, we tested mutations in the sequence UUUCAA/A at the 3' splice site of the first intron of a test gene in vivo. Replacement of individual U's with A's or C's compromised splicing. When the sequence UUUCAA/AAG was tested, splicing occurred following both the -1A and the +3G, indicating that both UUUC and the AG contain 3' splice site information. When the sequence UUUCAA/AAA was tested, all splicing occurred following the -1A, suggesting that the UUUC contains sufficient information in the absence of an AG to specify the location of the splice site. In support of this idea, when point mutations were introduced into the UUUC, unspliced RNAs accumulated. Surprisingly, RNAs containing the mutant intron often contained the second, nonmutated intron as well, suggesting that interference with splicing of one intron can interfere with splicing of a second intron in the same pre-mRNA. The majority of these unspliced RNAs were degraded by the system responsible for degradation of transcripts containing nonsense mutations (smg), even though the intron contained no nonsense codon.

Animals↗