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S M Berget

Publications and source records attributed to S M Berget.

At least 19 recordsLinked to original sources
PubMed1999

Stage-specific changes in SR splicing factors and alternative splicing in mammary tumorigenesis.

Using a mouse model of mammary gland development and tumorigenesis we examined changes in both alternative splicing and splicing factors in multiple stages of mammary cancer. The emphasis was on the SR family of splicing factors known to influence alternative splicing in a wide variety of genes, and on alternative splicing of the pre-mRNA encoding CD44, for which alternative splicing has been implicated as important in a number of human cancers, including breast cancer. We observed step-wise increases in expression of individual SR proteins and alternative splicing of CD44 mRNA during mammary gland tumorigenesis. Individual preneoplasias differed as to their expression patterns for SR proteins, often expressing only a sub-set of the family. In contrast, tumors demonstrated a complex pattern of SR expression. Little difference was observed between neoplasias and their metastases. Alternative splicing of CD44 also changed through the disease paradigm such that tumors produced RNA containing a mixture of variable exons, whereas preneoplasias exhibited a more restricted exon inclusion pattern. In contrast, other standard splicing factors changed little in either concentration or splicing pattern in the same cells. These data suggest alterations in relative concentrations of specific splicing factors during early preneoplasia that become more pronounced during tumor formation. Given the ability of SR proteins to affect alternative processing decisions, our results suggest that a number of pre-mRNAs may undergo changes in alternative splicing during the early and intermediate stages of mammary cancer.

Alternative Splicing↗
PubMed1999

Polypyrimidine tract-binding protein positively regulates inclusion of an alternative 3'-terminal exon.

Polypyrimidine tract-binding protein (PTB) is an abundant vertebrate hnRNP protein. PTB binding sites have been found within introns both upstream and downstream of alternative exons in a number of genes that are negatively controlled by the binding of PTB. We have previously reported that PTB binds to a pyrimidine tract within an RNA processing enhancer located adjacent to an alternative 3'-terminal exon within the gene coding for calcitonin and calcitonin gene-related peptide. The enhancer consists of a pyrimidine tract and CAG directly abutting on a 5' splice site sequence to form a pseudoexon. Here we show that the binding of PTB to the enhancer pyrimidine tract is functional in that exon inclusion increases when in vivo levels of PTB increase. This is the first example of positive regulation of exon inclusion by PTB. The binding of PTB was antagonistic to the binding of U2AF to the enhancer-located pyrimidine tract. Altering the enhancer pyrimidine tract to a consensus sequence for the binding of U2AF eliminated enhancement of exon inclusion in vivo and exon polyadenylation in vitro. An additional PTB binding site was identified close to the AAUAAA hexanucleotide sequence of the exon 4 poly(A) site. These observations suggest a dual role for PTB in facilitating recognition of exon 4: binding to the enhancer pyrimidine tract to interrupt productive recognition of the enhancer pseudoexon by splicing factors and interacting with the poly(A) site to positively affect polyadenylation.

Animals↗
PubMed1998

A short sequence within two purine-rich enhancers determines 5' splice site specificity.

Purine-rich enhancers are exon sequences that promote inclusion of alternative exons, usually via activation of weak upstream 3' splice sites. A recently described purine-rich enhancer from the caldesmon gene has an additional activity by which it directs selection of competing 5' splice sites within an alternative exon. In this study, we have compared the caldesmon enhancer with another purine-rich enhancer from the chicken cardiac troponin T (cTNT) gene for the ability to regulate flanking splice sites. Although similar in sequence and length, the two enhancers demonstrated strikingly different specificities towards 5' splice site choice when placed between competing 5' splice sites in an internal exon. The 32-nucleotide caldesmon enhancer caused effective usage of the exon-internal 5' splice site, whereas the 30-nucleotide cTNT enhancer caused effective usage of the exon-terminal 5' splice site. Both enhancer-mediated splicing pathways represented modulation of the default pathway in which both 5' splice sites were utilized. Each enhancer is multipartite, consisting of two purine-rich sequences of a simple (GAR)n repeat interdigitated with two enhancer-specific sequences. The entire enhancer was necessary for maximal splice site selectivity; however, a 5- to 7-nucleotide region from the 3' end of each enhancer dictated splice site selectivity. Mutations that interchanged this short region of the two enhancers switched specificity. The portion of the cTNT enhancer determinative for 5' splice site selectivity was different than that shown to be maximally important for activation of a 3' splice site, suggesting that enhancer environment can have a major impact on activity. These results are the first indication that individual purine-rich enhancers can differentiate between flanking splice sites. Furthermore, localization of the specificity of splice site choice to a short region within both enhancers indicates that subtle differences in enhancer sequence can have profound effects on the splicing pathway.

Animals↗
PubMed1998

Regulation of alternative polyadenylation by U1 snRNPs and SRp20.

Although considerable information is currently available about the factors involved in constitutive vertebrate polyadenylation, the factors and mechanisms involved in facilitating communication between polyadenylation and splicing are largely unknown. Even less is known about the regulation of polyadenylation in genes in which 3'-terminal exons are alternatively recognized. Here we demonstrate that an SR protein, SRp20, affects recognition of an alternative 3'-terminal exon via an effect on the efficiency of binding of a polyadenylation factor to an alternative polyadenylation site. The gene under study codes for the peptides calcitonin and calcitonin gene-related peptide. Its pre-mRNA is alternatively processed by the tissue-specific inclusion or exclusion of an embedded 3'-terminal exon, exon 4, via factors binding to an intronic enhancer element that contains both 3' and 5' splice site consensus sequence elements. In cell types that preferentially exclude exon 4, addition of wild-type SRp20 enhances exon 4 inclusion via recognition of the intronic enhancer. In contrast, in cell types that preferentially include exon 4, addition of a mutant form of SRp20 containing the RNA-binding domain but missing the SR domain inhibits exon 4 inclusion. Inhibition is likely at the level of polyadenylation, because the mutant SRp20 inhibits binding of CstF to the exon 4 poly(A) site. This is the first demonstration that an SR protein can influence alternative polyadenylation and suggests that this family of proteins may play a role in recognition of 3'-terminal exons and perhaps in the communication between polyadenylation and splicing.

Animals↗
PubMed1998

A role for SRp54 during intron bridging of small introns with pyrimidine tracts upstream of the branch point.

One of the earliest steps in pre-mRNA recognition involves binding of the splicing factor U2 snRNP auxiliary factor (U2AF or MUD2 in Saccharomyces cerevisiae) to the 3' splice site region. U2AF interacts with a number of other proteins, including members of the serine/arginine (SR) family of splicing factors as well as splicing factor 1 (SF1 or branch point bridging protein in S. cerevisiae), thereby participating in bridging either exons or introns. In vertebrates, the binding site for U2AF is the pyrimidine tract located between the branch point and 3' splice site. Many small introns, especially those in nonvertebrates, lack a classical 3' pyrimidine tract. Here we show that a 59-nucleotide Drosophila melanogaster intron contains C-rich pyrimidine tracts between the 5' splice site and branch point that are needed for maximal binding of both U1 snRNPs and U2 snRNPs to the 5' and 3' splice site, respectively, suggesting that the tracts are the binding site for an intron bridging factor. The tracts are shown to bind both U2AF and the SR protein SRp54 but not SF1. Addition of a strong 3' pyrimidine tract downstream of the branch point increases binding of SF1, but in this context, the upstream pyrimidine tracts are inhibitory. We suggest that U2AF- and/or SRp54-mediated intron bridging may be an alternative early recognition mode to SF1-directed bridging for small introns, suggesting gene-specific early spliceosome assembly.

Amino Acid Sequence↗
PubMed1997

Dynamic relocation of transcription and splicing factors dependent upon transcriptional activity.

Recent interest in understanding the spatial organization of gene expression has focused attention on nuclear structures known as speckles or interchromatin granule clusters (IGCs) revealed by immunofluorescence or electron microscopy. Staining of nuclear factors involved in pre-mRNA splicing or, more recently, transcription, reveals 20-40 speckles per nucleus, resulting in the intriguing suggestion that speckles are nuclear sites of transcription and processing. In contrast, other investigations have observed transcription in other areas of the nucleus. In this study, we have examined the localization of active transcription as detected by uridine incorporation and recently developed RNA polymerase II antibodies, and compared this pattern with that of known splicing and polyadenylation factors. Our results indicate that in actively transcribing cells, transcription and splicing factors are dispersed throughout the nucleus with abundant sites of preferred localization. In contrast, in poorly transcribing cells, polymerase II and splicing factors localize to speckles. In nuclei inactivated for transcription by drugs or heat shock, the speckle type of co-localization is accentuated. These observations suggest that bulk transcription and splicing occur throughout the nucleus during periods of active transcription; and that factors involved in these two processes re-locate to minimal speckle domains during periods of inactive transcription.

Animals↗
PubMed1997

A naturally arising mutation of a potential silencer of exon splicing in human immunodeficiency virus type 1 induces dominant aberrant splicing and arrests virus production.

We have isolated a naturally arising human immunodeficiency type 1 (HIV-1) mutant containing a point mutation within the env gene. The point mutation resulted in complete loss of balanced splicing, with dominant production of aberrant mRNAs. The aberrant RNAs arose via activation of normally cryptic splice sites flanking the mutation within the env terminal exon to create exon 6D, which was subsequently incorporated in aberrant env, tat, rev, and nef mRNAs. Aberrant multiply spliced messages contributed to reduced virus replication as a result of a reduction in wild-type Rev protein. The point mutation within exon 6D activated exon 6D inclusion when the exon and its flanking splice sites were transferred to a heterologous minigene. Introduction of the point mutation into an otherwise wild-type HIV-1 proviral clone resulted in virus that was severely inhibited for replication in T cells and displayed elevated usage of exon 6D. Exon 6D contains a bipartite element similar to that seen in tat exon 3 of HIV-1, consisting of a potential exon splicing silencer (ESS) juxtaposed to a purine-rich sequence similar to known exon splicing enhancers. In the absence of a flanking 5' splice site, the point mutation within the exon 6D ESS-like element strongly activated env splicing, suggesting that the putative ESS plays a natural role in limiting the level of env splicing. We propose, therefore, that exon silencers may be a common element in the HIV-1 genome used to create balanced splicing of multiple products from a single precursor RNA.

Alternative Splicing↗
PubMed1997

Pyrimidine tracts between the 5' splice site and branch point facilitate splicing and recognition of a small Drosophila intron.

The minimum size for splicing of a vertebrate intron is approximately 70 nucleotides. In Drosophila melanogaster, more than half of the introns are significantly below this minimum yet function well. Such short introns often lack the pyrimidine tract located between the branch point and 3' splice site common to metazoan introns. To investigate if small introns contain special sequences that facilitate their recognition, the sequences and factors required for the splicing of a 59-nucleotide intron from the D. melanogaster mle gene have been examined. This intron contains only a minimal region of interrupted pyrimidines downstream of the branch point. Instead, two longer, uninterrupted C-rich tracts are located between the 5' splice site and branch point. Both of these sequences are required for maximal in vivo and in vitro splicing. The upstream sequences are also required for maximal binding of factors to the 5' splice site, cross-linking of U2AF to precursor RNA, and assembly of the active spliceosome, suggesting that sequences upstream of the branch point influence events at both ends of the small mle intron. Thus, a very short intron lacking a classical pyrimidine tract between the branch point and 3' splice site requires accessory pyrimidine sequences in the short region between the 5' splice site and branch point.

Animals↗
PubMed1997

G triplets located throughout a class of small vertebrate introns enforce intron borders and regulate splice site selection.

Splicing of small introns in lower eucaryotes can be distinguished from vertebrate splicing by the inability of such introns to be expanded and by the inability of splice site mutations to cause exon skipping-properties suggesting that the intron rather than the exon is the unit of recognition. Vertebrates do contain small introns. To see if they possess properties similar to small introns in lower eucaryotes, we studied the small second intron from the human alpha-globin gene. Mutation of the 5' splice site of this intron resulted in in vivo intron inclusion, not exon skipping, suggesting the presence of intron bridging interactions. The intron had an unusual base composition reflective of a sequence bias present in a collection of small human introns in which multiple G triplets stud the interior of the introns. Each G triplet represented a minimal sequence element additively contributing to maximal splicing efficiency and spliceosome assembly. More importantly, G triplets proximal to a duplicated splice site caused preferential utilization of the 5' splice site upstream of the triplets or the 3' splice site downstream of the triplets; i.e., sequences containing G triplets were preferentially used as introns when a choice was possible. Thus, G triplets internal to a small intron have the ability to affect splice site decisions at both ends of the intron. Each G triplet additively contributed to splice site selectivity. We suggest that G triplets are a common component of human 5' splice sites and aid in the definition of exon-intron borders as well as overall splicing efficiency. In addition, our data suggest that such intronic elements may be characteristic of small introns and represent an intronic equivalent to the exon enhancers that facilitate recognition of both ends of an exon during exon definition.

Animals↗
PubMed1996

Architectural limits on split genes.

Exon/intron architecture varies across the eukaryotic kingdom with large introns and small exons the rule in vertebrates and the opposite in lower eukaryotes. To investigate the relationship between exon and intron size in pre-mRNA processing, internally expanded exons were placed in vertebrate genes with small and large introns. Both exon and intron size influenced splicing phenotype. Intron size dictated if large exons were efficiently recognized. When introns were large, large exons were skipped; when introns were small, the same large exons were included. Thus, large exons were incompatible for splicing if and only if they were flanked by large introns. Both intron and exon size became problematic at approximately 500 nt, although both exon and intron sequence influenced the size at which exons and introns failed to be recognized. These results indicate that present-day gene architecture reflects at least in part limitations on exon recognition. Furthermore, these results strengthen models that invoke pairing of splice sites during recognition of pre-mRNAs, and suggest that vertebrate consensus sequences support pairing across either introns or exons.

Adenine Phosphoribosyltransferase↗
PubMed1996

An intron enhancer recognized by splicing factors activates polyadenylation.

Alternative processing of the pre-messenger RNA encoding calcitonin/calcitonin gene-related peptide (CT/CGRP) involves alternative inclusion of a 3'-terminal exon (exon 4) embedded within a six exon primary transcript. Expression of CT/CGRP in transgenic mice indicates that inclusion of exon 4 occurs in a wide variety of tissues, suggesting that the factors responsible for exon 4 inclusion are widely distributed. Inclusion of exon 4 requires an enhancer sequence located within the intron downstream of the poly(A) site of exon 4. Here we show that the intron enhancer activated in vitro polyadenylation cleavage of precursor RNAs containing the CT/CGRP exon 4 poly(A) site or heterologous poly(A) sites. To our knowledge this is the first example of an intron-located enhancer that facilitates polyadenylation. Within the enhancer sequence is a 5' splice site sequence immediately preceded by a pyrimidine tract. This 5' splice site sequence was required for enhanced polyadenylation and was recognized by both U1 small nuclear ribonucleoproteins (snRNPs) and alternative splicing factor/splicing factor 2 (ASF/SF2). Enhancement of polyadenylation required U1 RNA, suggesting that the 5' splice site sequence within the enhancer mediates enhancement via interaction with factors normally associated with functional 5' splice sites. Mutation of the polypyrimidine track of the enhancer also inhibited in vitro polyadenylation cleavage. Oligonucleotide competitions and UV cross-linking indicated that the enhancer pyrimidine track binds the polypyrimidine tract binding protein (PTB), but not U2 snRNP auxiliary factor (U2AF), and that binding of PTB was required for maximal enhancer-mediated polyadenylation. These results suggest that the enhancer binds known splicing factors, and that binding of these factors activates polyadenylation cleavage. Furthermore, these results suggest that regulation of alternative processing of CT/CGRP could occur at the level of polyadenylation, rather than splicing.

Alternative Splicing↗
PubMed1996

An intron splicing enhancer containing a G-rich repeat facilitates inclusion of a vertebrate micro-exon.

The average length of a vertebrate axon is approximately 130 nt. Decreasing the size of an internal axon to less than 51 nt induces axon skipping, implying a minimal size for exons. A few constitutively included internal exons, however, are extremely small. To investigate if such micro-exons require special mechanisms for their inclusion, we studied the sequences necessary for inclusion of a 6-nt axon from chicken cardiac troponin T (cTNT). In vivo, the cTNT micro-exon was not included in mRNA unless accompanied by a 134-nt sequence located next to the micro-exon in the downstream intron. Increasing the length of the micro-exon alleviated the requirement for the intron element, indicating that the lack of inclusion of the micro-exon in the absence of a facilitating sequence was due to its small size, rather than suboptimal splice sites. The intron element contained six copies of a G-rich 7-nt sequence. Multimers of the repeat supported exon inclusion, indicating that the repeat sequence is an important part of the intron element. The entire intron element activated inclusion of a heterologous 7-nt exon, suggesting that the intron element is a general enhancer for the splicing of micro-exons. In vitro, the intron element and the repeated sequence facilitated splicing of a heterologous exon. Because of the ability of the cTNT intron element to facilitate the splicing of heterologous exons, we have termed the element an intron splicing enhancer (ISE). Interestingly, the ISE demonstrated position independence in that it facilitated inclusion of the heterologous micro-exon when placed either upstream or downstream of the micro-exon. In vitro, the ISE or copies of the ISE G-rich repeat stimulated splicing of an adjacent intron. The ISE thus becomes one of only a few characterized ISEs containing a G-rich repeat and the first to work both upstream and downstream of a target axon.

3T3 Cells↗
PubMed1995

An intron enhancer containing a 5' splice site sequence in the human calcitonin/calcitonin gene-related peptide gene.

Regulation of calcitonin (CT)/calcitonin gene-related peptide (CGRP) RNA processing involves the use of alternative 3' terminal exons. In most tissues and cell lines, the CT terminal exon is recognized. In an attempt to define regulatory sequences involved in the utilization of the CT-specific terminal exon, we performed deletion and mutation analyses of a mini-gene construct that contains the CT terminal exon and mimics the CT processing choice in vivo. These studies identified a 127-nucleotide intron enhancer located approximately 150 nucleotides downstream of the CT exon poly(A) cleavage site that is required for recognition of the exon. The enhancer contains an essential and conserved 5' splice site sequence. Mutation of the splice site resulted in diminished utilization of the CT-specific terminal exon and increased skipping of the CT exon in both the mini-gene and in the natural CT/CGRP gene. Other components of the intron enhancer modified utilization of the CT-specific terminal exon and were necessary to prevent utilization of the 5' splice site within the intron enhancer as an actual splice site directing cryptic splicing. Conservation of the intron enhancer in three mammalian species suggests an important role for this intron element in the regulation of CT/CGRP processing and an expanded role for intronic 5' splice site sequences in the regulation of RNA processing.

Alternative Splicing↗
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↗