Biomedical subjects

S M Berget

Publications and source records attributed to S M Berget.

At least 37 records · Page 2Linked to original sources

PubMed1995

A 32-nucleotide exon-splicing enhancer regulates usage of competing 5' splice sites in a differential internal exon.

Large alternatively spliced internal exons are uncommon in vertebrate genes, and the mechanisms governing their usage are unknown. In this report, we examined alternative splicing of a 1-kb internal exon from the human caldesmon gene containing two regulated 5' splice sites that are 687 nucleotides apart. In cell lines normally splicing caldesmon RNA via utilization of the exon-internal 5' splice site, inclusion of the differential exon required a long purine-rich sequence located between the two competing 5' splice sites. This element consisted of four identical 32-nucleotide purine-rich repeats that resemble exon-splicing enhancers (ESE) identified in other genes. One 32-nucleotide repeat supported exon inclusion, repressed usage of the terminal 5' splice site, and functioned in a heterologous exon dependent on exon enhancers for inclusion, indicating that the caldesmon purine-rich sequence can be classified as an ESE. The ESE was required for utilization of the internal 5' splice site only in the presence of the competing 5' splice site and had no effect when placed downstream of the terminal 5' splice site. In the absence of the internal 5' splice site, the ESE activated a normally silent cryptic 5' splice site near the natural internal 5' splice site, indicating that the ESE stimulates upstream 5' splice site selection. We propose that the caldesmon ESE functions to regulate competition between two 5' splice sites within a differential internal exon.

Alternative Splicing↗

PubMed1994

Nuclear-mitotic apparatus protein: a structural protein interface between the nucleoskeleton and RNA splicing.

Vertebrate splicing factors are localized to discrete domains within the nuclei of somatic cells. The mechanism whereby such nuclear domains, identified as speckles by immunofluorescence microscopy, are generated is unclear. Recent studies suggest that the spatial order within the nucleus is maintained by nuclear matrix factors. Here we show that a protein in the nuclear matrix and mitotic apparatus [nuclear-mitotic apparatus protein, NuMA; Lydersen, B. & Pettijohn, D. (1980) Cell 22, 489-499] colocalizes with splicing factors in interphase nuclei and is associated with small nuclear ribonucleoproteins in a complex immunoprecipitated from HeLa extract with small nuclear ribonucleoprotein antibodies. Moreover, NuMA associates with splicing complexes that are reconstituted in vitro using wild-type pre-mRNA, but not with nonspecific RNA. Cumulatively, these observations suggest a function of NuMA or NuMA-like proteins in interphase cells in providing a bridge between RNA processing and the nucleoskeleton.

Antigens, Nuclear↗

PubMed1994

Intron definition in splicing of small Drosophila introns.

Approximately half of the introns in Drosophila melanogaster are too small to function in a vertebrate and often lack the pyrimidine tract associated with vertebrate 3' splice sites. Here, we report the splicing and spliceosome assembly properties of two such introns: one with a pyrimidine-poor 3' splice site and one with a pyrimidine-rich 3' splice site. The pyrimidine-poor intron was absolutely dependent on its small size for in vivo and in vitro splicing and assembly. As such, it had properties reminiscent of those of yeast introns. The pyrimidine-rich intron had properties intermediate between those of yeasts and vertebrates. This 3' splice site directed assembly of ATP-dependent complexes when present as either an intron or exon and supported low levels of in vivo splicing of a moderate-length intron. We propose that splice sites can be recognized as pairs across either exons or introns, depending on which distance is shorter, and that a pyrimidine-rich region upstream of the 3' splice site facilitates the exon mode.

Animals↗

PubMed1993

Efficient selection of 3'-terminal exons from vertebrate DNA.

Identification of expressed sequences within genomic DNA is a hurdle in the characterization of complex genomes. We developed an exon trapping scheme that provides a positive selection for vertebrate 3'-terminal exons. A copy of the trapped exon sequence is obtained by RT/PCR amplification. The technique detects valid terminal exons without interference from partial exons or non-specific sequences, including simple human repeated sequences. Application to random human cosmids yielded one unique trapped terminal exon per cosmid on average. Because vertebrate terminal exons average 600-700 nucleotides in length, the technique provides transcribed sequences of sufficient length to assist further mapping efforts.

Animals↗

PubMed1993

In vivo recognition of a vertebrate mini-exon as an exon-intron-exon unit.

Very small vertebrate exons are problematic for RNA splicing because of the proximity of their 3' and 5' splice sites. In this study, we investigated the recognition of a constitutive 7-nucleotide mini-exon from the troponin I gene that resides quite close to the adjacent upstream exon. The mini-exon failed to be included in spliced RNA when placed in a heterologous gene unless accompanied by the upstream exon. The requirement for the upstream exon disappeared when the mini-exon was internally expanded, suggesting that the splice sites bordering the mini-exon are compatible with those of other constitutive vertebrate exons and that the small size of the exon impaired inclusion. Mutation of the 5' splice site of the natural upstream exon did not result in either exon skipping or activation of a cryptic 5' splice site, the normal vertebrate phenotypes for such mutants. Instead, a spliced RNA accumulated that still contained the upstream intron. In vitro, the mini-exon failed to assemble into spliceosome complexes unless either internally expanded or accompanied by the upstream exon. Thus, impaired usage of the mini-exon in vivo was accompanied by impaired recognition in vitro, and recognition of the mini-exon was facilitated by the presence of the upstream exon in vivo and in vitro. Cumulatively, the atypical in vivo and in vitro properties of the troponin exons suggest a mechanism for the recognition of this mini-exon in which initial recognition of an exon-intron-exon unit is followed by subsequent recognition of the intron.

3T3 Cells↗

PubMed1992

Are vertebrate exons scanned during splice-site selection?

Pairwise recognition of splice sites as a result of a scanning mechanism is an attractive model to explain the coordination of vertebrate splicing. Such a mechanism would predict a polarity-of-site recognition in the scanned unit, but no evidence for a polarity gradient across introns has been found. We have suggested that the exon rather than the intron is the unit of recognition in vertebrates and that polyadenylation and splicing factors interact during recognition of 3'-terminal exons. Interaction is reflected in maximal rates of in vitro polyadenylation. If scanning across the exon is operating during this interaction, then insertion of a 5' splice site should depress polyadenylation. Here we report recognition in vitro and in vivo of a 5' splice site situated within a 3'-terminal exon, and a concomitant depression of polyadenylation and ultraviolet crosslinking of a polyadenylation factor. Decreased crosslinking was only found when the 3' and 5' splice sites were within 300 nucleotides of each other. These results are consistent with an exon scanning mechanism for splice-site selection.

Adenoviridae↗

PubMed1992

Identification of exon sequences and an exon binding protein involved in alternative RNA splicing of calcitonin/CGRP.

Transcripts derived from the 6 exon CALC I gene are differentially processed in a tissue-specific fashion to include or exclude a calcitonin-specific exon 4. All cell types which transcribe a second calcitonin/CGRP gene, CALC II, exclude exon 4. Substitution of the first 30 nucleotides of CALC I exon 4 with analogous CALC II sequence was sufficient to prevent recognition of exon 4 in in vitro or in vivo RNA splicing systems. UV crosslinking detected a approximately 66 kDa RNA-binding protein in HeLa nuclear extract which interacted with CALC I proximal exon sequence, but not CALC II or mutant sequences. UV crosslinking of this protein was inhibited by addition of nuclear extract from a cell type which normally causes exclusion of exon 4. These results identify an important regulatory element within exon 4 and support a model in which calcitonin production requires protein interaction with this sequence to facilitate exon recognition.

Amino Acid Sequence↗

PubMed1991

Validation of an in vitro RNA processing system for CT/CGRP precursor mRNA.

The pre-mRNA encoding calcitonin (CT) and calcitonin gene-related peptide (CGRP) is differentially processed in a tissue-specific fashion to include or exclude the calcitonin-specific exon 4. A minigene containing a viral first exon and exons 4, 5, and 6 from the human CT/CGRP gene was correctly processed in transfected HeLa or F9 teratocarcinoma cells to produce mRNA that included or excluded exon 4, respectively. This processing decision could be reproduced in vitro using nuclear extracts from these two cell lines and an RNA precursor from a similar minigene. Supplementation of extract from HeLa cells with extract from F9 cells resulted in the F9 splicing pattern in which exon 4 was excluded. This model system may be useful for the purification of splicing factors important in the regulation of this splice choice.

Adenoviridae↗

PubMed1991

Identification of nuclear proteins that specifically bind to RNAs containing 5' splice sites.

Two polypeptides of 26 and 37 kDa (designated SPP-1 and SPP-2) were identified in in vitro splicing extracts by UV crosslinking to splicing precursor RNAs. Crosslinking of both polypeptides required a functional 5' splice site but was not dependent on sequences at the 3' end of the intron. Centrifugation of extract separated the two polypeptides from major U small nuclear ribonucleoproteins (snRNPs), including U1 snRNPs. Both polypeptides crosslinked to precursor RNAs containing 5' splice sites in the absence of U1 RNA. Complexes containing both polypeptides also contained U1 snRNPs, suggesting that SPP-1 and SPP-2 are a part of the functional spliceosome. We propose that SPP-1 and SPP-2 are factors that participate in the recognition of 5' splice sites.

Adenoviruses, Human↗

PubMed1991

Mutation of the AAUAAA polyadenylation signal depresses in vitro splicing of proximal but not distal introns.

To investigate the relationship between splicing and polyadenylation during the production of vertebrate mRNAs, we examined the effect of mutation of a poly(A) site on splicing of upstream introns. Mutation of the AAUAAA polyadenylation consensus sequence inhibited in vitro splicing of an upstream intron. The magnitude of the depression depended on the magnesium concentration. Dependence of splicing on polyadenylation signals suggests the existence of interaction between polyadenylation and splicing factors. In multi-intron precursor RNAs containing duplicated splice sites, mutation of the poly(A) site inhibited removal of the last intron, but not the removal of introns farther upstream. Inhibition of removal of only the last intron suggests segmental recognition of multi-exon precursor RNAs and is consistent with previous suggestions that signals at both ends of an exon are required for effective splicing of an upstream intron.

Adenoviridae↗

PubMed1991

Polyadenylation precedes splicing in vitro.

Vertebrate premessenger RNAs are usually spliced and polyadenylated. In vivo analysis of the relative kinetics of the two reactions is difficult. We have used in vitro processing systems to investigate the order of splicing and polyadenylation of chimeric precursor RNAs containing a single intron and a poly(A) site. Polyadenylated, but not spliced, intermediate RNA appeared first and reached a low steady-state level early during incubation, properties consistent with its being a reaction intermediate in the production of doubly-processed spliced and polyadenylated product RNA. The kinetics of polyadenylation suggested that polyadenylated RNA was the only intermediate in the production of doubly-processed RNA. Spliced, but not polyadenylated, RNA also appeared. This species, however, continued to accumulate during reaction, and could not be chased into product spliced and polyadenylated RNA. These data support a preferred order of reaction for 3' terminal introns and exons in which polyadenylation precedes splicing.

Adenoviridae↗

PubMed1990

In vitro polyadenylation is stimulated by the presence of an upstream intron.

The majority of vertebrate pre-mRNAs are both spliced and polyadenylated. To investigate the mechanism whereby processing factors recognize last exons containing both splicing and polyadenylation consensus elements, chimeric precursor RNAs containing a single intron and a poly(A) site were constructed and assayed for in vitro splicing and polyadenylation. Chimeric RNAs underwent splicing and polyadenylation. Both reactions occurred in a single RNA. The presence of an intron enhanced the rate of polyadenylation at a downstream poly(A) site. The extent of stimulation varied from two- to fivefold, depending on the magnesium concentration. Maximal stimulation of polyadenylation by an upstream intron required a 3' splice site but not a 5' splice site, suggesting that the structure of the terminal exon was more important than the presence of a complete upstream intron. We suggest that splicing and polyadenylation factors interact to recognize terminal, poly(A) site-containing exons. Such interaction may explain why all known intron-containing eukaryotic pre-mRNAs generate their 3' ends by polyadenylation.

Base Sequence↗

PubMed1990

Exon definition may facilitate splice site selection in RNAs with multiple exons.

Interactions at the 3' end of the intron initiate spliceosome assembly and splice site selection in vertebrate pre-mRNAs. Multiple factors, including U1 small nuclear ribonucleoproteins (snRNPs), are involved in initial recognition at the 3' end of the intron. Experiments were designed to test the possibility that U1 snRNP interaction at the 3' end of the intron during early assembly functions to recognize and define the downstream exon and its resident 5' splice site. Splicing precursor RNAs constructed to have elongated second exons lacking 5' splice sites were deficient in spliceosome assembly and splicing activity in vitro. Similar substrates including a 5' splice site at the end of exon 2 assembled and spliced normally as long as the second exon was less than 300 nucleotides long. U2 snRNPs were required for protection of the 5' splice site terminating exon 2, suggesting direct communication during early assembly between factors binding the 3' and 5' splice sites bordering an exon. We suggest that exons are recognized and defined as units during early assembly by binding of factors to the 3' end of the intron, followed by a search for a downstream 5' splice site. In this view, only the presence of both a 3' and a 5' splice site in the correct orientation and within 300 nucleotides of one another will stable exon complexes be formed. Concerted recognition of exons may help explain the 300-nucleotide-length maximum of vertebrate internal exons, the mechanism whereby the splicing machinery ignores cryptic sites within introns, the mechanism whereby exon skipping is normally avoided, and the phenotypes of 5' splice site mutations that inhibit splicing of neighboring introns.

Animals↗

PubMed1990

UV cross-linking of polypeptides associated with 3'-terminal exons.

Association of nuclear proteins with chimeric vertebrate precursor RNAs containing both polyadenylation signals and an intron was examined by UV cross-linking. One major difference in cross-linking pattern was observed between this chimeric precursor RNA and precursors containing only polyadenylation or splicing signals. The heterogeneous nuclear ribonucleoprotein (hnRNP) polypeptide C cross-linked strongly to sequences downstream of the A addition site in polyadenylation precursor RNA containing only the polyadenylation signal from the simian virus 40 (SV40) late transcription unit. In contrast, the hnRNP C polypeptide cross-linked to chimeric RNA containing the same SV40 late poly(A) cassette very poorly, at a level less than 5% of that observed with the precursor RNA containing just the poly(A) site. Observation that cross-linking of the hnRNP C polypeptide to elements within the SV40 late poly(A) site was altered by the presence of an upstream intron suggests differences in the way nuclear factors associate with poly(A) sites in the presence and absence of an upstream intron. Cross-linking of C polypeptide to chimeric RNA increased with RNAs mutated for splicing or polyadenylation consensus sequences and under reaction conditions (high magnesium) that inhibited polyadenylation. Furthermore, cross-linking of hnRNP C polypeptide to precursors containing just the SV40 late poly(A) site was eliminated in the presence of competing poly(U); polyadenylation, however, was unaffected. Correlation of loss of activity with high levels of hnRNP C polypeptide cross-linking raises questions about the specificity of the interaction between the hnRNP C polypeptide and polyadenylation precursor RNAs in vitro.

Animals↗

PubMed1990

Effect of 5' splice site mutations on splicing of the preceding intron.

Three exon constructs containing identical intron and exon sequences were mutated at the 5' splice site beginning intron 2 and assayed for the effect of the mutation on splicing of the upstream intron in vitro. Alteration of two or six bases within the 5' splice site reduced removal of intron 1 at least 20-fold, as determined by quantitation of either spliced product or released lariat RNA. The prominent product was skip splicing of exon 1 to exon 3. Examination of complex formation indicated that mutation of the 5' splice site terminating exon 2 depressed the ability of precursor RNAs containing just the affected exon to direct assembly in vitro. These results suggest that mutation at the end of an internal exon inhibits the ability of the exon to be recognized by splicing factors. A comparison of the known vertebrate 5' splice site mutations in which the mutation resides at the end of an internal exon indicated that exon skipping is the preferred phenotype for this type of mutation, in agreement with the in vitro observation reported here. Inhibition of splicing by mutation at the distal and of the exon supports the suggestion that exons, rather than splice sites, are the recognition units for assembly of the spliceosome.

Adenoviruses, Human↗

PubMed1990

Calcitonin exon sequences influence alternative RNA processing.

The pre-mRNA encoding calcitonin (CT) and CT gene-related peptide (CGRP) is differentially processed in a tissue-specific fashion to include exon 4 (which encodes CT) or exclude this exon and splice to exon 5 (which encodes CGRP). We have used a CT-specific in vitro RNA-processing system to identify cis-acting sequences required to prevent splicing to exon 5. Deletion mapping demonstrated the presence of an element within the first 45 nucleotides of the CT-specific exon 4 that was required to suppress splicing to the CGRP-specific exon 5. This element was able to function in a completely heterologous system to suppress splicing when the CGRP exon was replaced with a constitutive viral exon. The element was unable to suppress splicing in the absence of a proximal CT-specific 3' splice site. Our results suggest that CT-specific splicing requires assisted recognition of its 3' splice site.

Calcitonin↗

PubMed1989

Evidence for nuclear factors involved in recognition of 5' splice sites.

To investigate soluble factors involved in pre-messenger RNA splicing we have fractionated nuclear extract by simple centrifugation to produce a supernatant pellet pair. Factors larger than 15S including U2, U4, U5, and U6 snRNPs fractionate with the pellet; U1 snRNPs distribute equally in pellet and supernatant. Each fraction is individually incompetent for splicing and spliceosome assembly; mixing restores wild type activity and assembly. The pellet fraction directs an aberrant assembly pathway in which proper 3', but improper 5' splice site recognition occurs. Complexes formed with the pellet fraction are distinguishable from wild-type complexes using native gel electrophoresis. Pellet complexes contain U1 snRNP antigens and their formation requires ATP, U1 snRNPs, U2 snRNPs, and sequences at the 3' end of the intron - properties shared with the initial steps of normal assembly and directed by sequences at the 3' end of the intron. In contrast, pellet complex assembly shows no dependence on the presence of a 5' splice junction within precursor RNA. Furthermore, binding of factors to the 5' splice junction is deficient in pellet assemblies. Thus, the pellet lacks a factor required for proper recognition of 5' splice sites. This factor can be supplied by the supernatant. Complementation occurs when supernatant U1 RNA is destroyed, suggesting that the supernatant factor recognizing 5' splice sites is not U1 snRNPs.

Adenosine Triphosphate↗

PubMed1989

Evidence that a nuclear matrix protein participates in premessenger RNA splicing.

The role of nuclear matrix proteins in premessenger RNA splicing has been investigated using antibodies raised against isolated rat liver nuclear matrix and cross-reactive with a 65-kDa HeLa cell nuclear matrix protein (IGA-65). IGA-65 is an internal nuclear matrix component which can be solubilized as a component of nuclear splicing extracts, by the action of endogenous ribonucleases, EDTA, and DTT during extract preparation. Preincubation of splicing extract with antibodies against IGA-65 (anti-IGA-65) inhibited in vitro splicing of exogenous adenovirus precursor RNA. Furthermore, assembly of precursor RNA into active spliceosome complexes was inhibited by pretreatment of extracts with anti-IGA-65, suggesting a role for IGA-65 during early spliceosome assembly. The IGA-65 present in splicing extracts was distinguishable from known U-snRNP and hnRNP proteins on protein gels. Furthermore, electrophoresis of splicing extract on native gels indicated that IGA-65 was present in protein complexes different from those containing U-snRNPs or hnRNP C protein. The data support identification of complexes containing IGA-65 as nuclear factors involved in pre-mRNA splicing and, by extension, suggest a role for the nuclear matrix during processing in vivo.

Antibodies↗