Alternative Splicing Pathways Research Articles

N-terminal amino acid sequences of three functionally different troponin T isoforms from rabbit fast skeletal muscle

The different isoforms of fast skeletal muscle troponin T (TnT) are generated by alternative splicing of several 5′ exons in the fast TnT gene. In rabbit skeletal muscle this process results in three major fast TnT species, TnT 1f, TnT 2f and TnT 3f, that differ in a region of 30 to 40 amino acid residues near the N terminus. Differential expression of these three isoforms modulates the activation of the thin filament by calcium. To establish a basis for further structure-function studies, we have sequenced the N-terminal region of these proteins. TnT 2f is the fast TnT sequenced by Pearlstone et al.. The larger species TnT 1f contains six additional amino acid residues identical in sequence and position to those encoded by exon 4 in the rat fast skeletal muscle TnT gene. TnT 3f also contains that sequence but lacks 17 amino acid residues spanning the region encoded by exons 6 and 7 of the rat gene. These three TnTs appear to be generated by discrete alternative splicing pathways, each differing by a single event. Comparison of these TnT sequences with those from chicken fast skeletal muscle and bovine heart shows that the splicing pattern resulting in the excision of exon 4 is evolutionarily conserved and leads to a more calcium-sensitive thin filament.

Expression of the SmB′ splicing protein in rodent cells capable of following an alternative RNA splicing pathway

The expression of the SmB and SmB′ spliceosome proteins in a variety of cell types and tissues has been investigated. Although SmB is found in all cells studied, the SmB′ protein is found only in a small number of rodent cell types. The presence of this protein is correlated with the ability to utilize an alternative pathway of RNA splicing which is not available in most cell types. This is the first demonstration of tissue specific expression of a protein component of the spliceosome and suggests a role for SmB′ in the regulation of some cases of alternative RNA splicing.

Sequence, structure and evolution of the gene coding for sn-glycerol-3-phosphate dehydrogenase inDrosophila melanogaster

We present the complete nucleotide and deduced amino acid sequence for the gene encoding Drosophila sn-glycerol-3-phosphate dehydrogenase. A transcription unit of 5kb was identified which is composed of eight protein encoding exons. Three classes of transcripts were shown to differ only in the 3'-end and to code for three protein isoforms each with a different C-terminal amino acid sequence. Each transcript is shown to arise through the differential expression of three isotype-specific exons at the 3'-end of the gene by a developmentally regulated process of 3'-end formation and alternate splicing pathways of the pre-mRNA. In contrast, the 5'-end of the gene is simple in structure and each mRNA is transcribed from the same promoter sequence. A comparison of the organization of the Drosophila and murine genes and the primary amino acid sequence between a total of four species indicates that the GPDH gene-enzyme system is highly conserved and is evolving slowly.

Drosophila sn-glycerol-3-phosphate dehydrogenase isozymes are generated by alternate pathways of RNA processing resulting in different carboxyl-terminal amino acid sequences.

Glycerol-3-phosphate dehydrogenase (GPDH, Ec 1.1.1.8) in Drosophila melanogaster consists of a family of three isozymes designated as GPDH-1, 2, and 3 which exhibit a unique temporal and tissue-specific pattern of expression. While each isozyme is encoded by the same structural gene, they differ by the amino acid sequence at the COOH-terminal end, with GPDH-3 having the sequence Asn-His-Pro-Glu-His-Met-COOH and with GPDH-1 extended by the three amino acid sequence Glu-Asn-Leu-COOH. We have isolated both genomic and cDNA clones in order to examine the structure of the 3'-end of this gene and its transcriptional products. This analysis has demonstrated three classes of transcripts, each differing in the 3'-untranslated region and coding for an enzyme with a different COOH-terminal amino acid sequence. Each transcript is shown to arise through the differential expression of three isotype-specific exons at the 3'-end of the gene. We propose a model where the expression of each isotype-specific transcript is controlled through a developmentally regulated process of 3'-end formation and alternate splicing pathways of the pre-mRNA. Furthermore, since each transcript and its cognant isozyme is tissue-specific in expression, this model suggests a role for tissue-specific trans-acting factors in these processing events.

Structure of the human tyrosine hydroxylase gene: alternative splicing from a single gene accounts for generation of four mRNA types.

Tyrosine hydroxylase (TH) is a rate-limiting enzyme for catecholamine biosynthesis. Recently, Grima et al. (Nature (1987) 326, 707-711) and we (Biochem. Biophys. Res. Commun. (1987) 146, 971-975; Nucleic Acids Res. (1987) 15, 6733) reported four similar but distinct mRNAs that encode human TH. These mRNAs are constant for the major part, but are distinguishable from one another as to the insertion/deletion of 12-bp and 81-bp sequences near the N-terminus. We isolated genomic clones encoding the human TH gene and determined the nucleotide sequence. The human TH gene is split into 14 exons. The 12-bp insertion sequence is encoded by the 3'-terminal portion of the first exon. The 81-bp insertion sequence corresponds to the second exon. Taking into consideration also the results of Southern blot analysis of human genomic DNA, we concluded that the four types of human TH mRNA are produced through alternative splicing from a single gene. Two kinds of alternative splicing are involved: the alternative use of two donor sites in the first exon, and the inclusion/exclusion of the second exon. We propose a possible secondary structure for the latter alternative splicing pathway.

Factors influencing alternative splice site utilization in vivo.

To study factors that influence the choice of alternative pre-mRNA splicing pathways, we introduced plasmids expressing either wild-type or mutated simian virus 40 (SV40) early regions into tissue culture cells and then measured the quantities of small-t and large-T RNAs produced. One important element controlling splice site selection was found to be the size of the intron removed in the production of small-t mRNA; expansion of this intron (from 66 to 77 or more nucleotides) resulted in a substantial increase in the amount of small-t mRNA produced relative to large-T mRNA. This suggests that in the normal course of SV40 early pre-mRNA processing, large-T splicing is at a competitive advantage relative to small-t splicing because of the small size of the latter intron. Several additional features of the pre-mRNA that can influence splice site selection were also identified by analyzing the effects of mutations containing splice site duplications. These include the strengths of competing 5' splice sites and the relative positions of splice sites in the pre-mRNA. Finally, we showed that the ratio of small-t to large-T mRNA was 10 to 15-fold greater in human 293 cells than in HeLa cells or other mammalian cell types. These results suggest the existence of cell-specific trans-acting factors that can dramatically alter the pattern of splice site selection in a pre-mRNA.

Factors influencing alternative splice site utilization in vivo.

To study factors that influence the choice of alternative pre-mRNA splicing pathways, we introduced plasmids expressing either wild-type or mutated simian virus 40 (SV40) early regions into tissue culture cells and then measured the quantities of small-t and large-T RNAs produced. One important element controlling splice site selection was found to be the size of the intron removed in the production of small-t mRNA; expansion of this intron (from 66 to 77 or more nucleotides) resulted in a substantial increase in the amount of small-t mRNA produced relative to large-T mRNA. This suggests that in the normal course of SV40 early pre-mRNA processing, large-T splicing is at a competitive advantage relative to small-t splicing because of the small size of the latter intron. Several additional features of the pre-mRNA that can influence splice site selection were also identified by analyzing the effects of mutations containing splice site duplications. These include the strengths of competing 5' splice sites and the relative positions of splice sites in the pre-mRNA. Finally, we showed that the ratio of small-t to large-T mRNA was 10 to 15-fold greater in human 293 cells than in HeLa cells or other mammalian cell types. These results suggest the existence of cell-specific trans-acting factors that can dramatically alter the pattern of splice site selection in a pre-mRNA.

Splice commitment dictates neuron-specific alternative RNA processing in calcitonin/CGRP gene expression

The calcitonin/CGRP gene is a complex transcription unit in which developmentally regulated cell-specific alternative RNA processing results in the production of CGRP mRNA in neurons, and of calcitonin mRNA in thyroid C cells. Alternative poly(A) site selection and exon splicing lead to the production of these alternative mature transcripts. The wild-type and a series of mutated calcitonin/CGRP genes were expressed in heterologous cell types, which produced predominately calcitonin or CGRP mRNA. The results of these studies suggest that neurons express machinery or a factor that determines a specific alternative splicing pathway and consequently its associated alternative poly(A) site selection. It is hypothesized that a splice commitment regulatory factor might modulate pre-mRNA secondary structure, revealing a cryptic splice site required to generate CGRP mRNA in the brain.

Secretion of HLA-A and -B antigens via an alternative RNA splicing pathway.

Human class I major histocompatibility antigens (HLA-A, -B and -C) are integral membrane protein heterodimers, which are anchored in the membrane via a stretch of hydrophobic amino acids near the carboxyl terminus of the heavy chain. It has previously been shown that a mutagenized cell line secretes a water soluble form of the HLA-A2 antigen, due to a pattern of RNA splicing that removes exon 5 (encoding the transmembrane hydrophobic amino acids) from mature, HLA-A2--encoding transcripts. The present study was undertaken to assess whether a similar process might be operative in nonmutagenized cells. It is shown that water soluble class I molecules (primarily HLA-A24) are secreted by the T leukemic cell line HPB-ALL, and that alternative splicing removes exon 5 from a fraction of HLA-A24--encoding transcripts. It is further shown that class I molecules are secreted, possibly in an allele-specific fashion, from a variety of tumor cells and normal cells. The possible relationship between these findings and previous reports of HLA-A and -B antigens in human serum is discussed.

Generation of troponin T isoforms by alternative RNA splicing in avian skeletal muscle. Conserved and divergent features in birds and mammals.

We describe the isolation and sequence analysis of quail muscle cDNA clones encoding two closely related isoforms of the striated muscle contractile protein, troponin T. The cDNAs represent two troponin T mRNAs that exhibit an unusual sequence relationship. The two mRNAs have identical sequences over hundreds of nucleotides including 3' untranslated regions, but they differ dramatically in a discrete, internally located block of 38 nucleotides. The two alternative sequences of this 38-nucleotide block encode two different but related versions of amino acid residues 230-242, near the C terminus of the protein. These results are consistent with a novel mechanism of troponin T isoform generation by alternative mRNA splicing pathways from a single gene containing two different exons corresponding to amino acids 229-242, as recently proposed by Medford et al. (Medford, R. M., Nguyen, H. T., Destree, A. T., Summers, E., and Nadal-Ginard, B. (1984) Cell 38, 409-421). This proposal was based on analysis of a rat troponin T genomic DNA clone and a cDNA clone corresponding to one of the two alternatively spliced mRNAs. Our analysis of quail troponin T cDNA clones, apparently corresponding to two alternatively spliced mRNA species, provides important new evidence for this novel mechanism of troponin T isoform generation and reveals the differential splicing mechanism to be of great antiquity, antedating the bird-mammal divergence. One of the quail alternative isoform sequences clearly corresponds to one of the rat sequences, but the other quail alternative sequence does not correspond to either of the rat sequences. This result suggests a greater complexity of troponin T gene structure or a greater diversity of troponin T isoform genes than is currently known, and also has implications for the functional significance of the troponin T protein isoform heterogeneity. Comparison of quail and mammal alternative isoform sequences also reveals strongly conserved features which suggest that all the isoform alternative amino acid sequences are variations on a common structural theme.

Immunoglobulin RNA Rearrangements in B Lymphocyte Differentiation

Publisher Summary DNA rearrangements generate transcriptionally active light and heavy chain genes. These DNA rearrangements are unique among the higher eukaryotic genes studied to date. In conjunction with multiple germline, variable region genes and yet unresolved mechanisms that generate somatic mutations in variable regions, these DNA joining events contribute significantly to the immense diversity of antibodies. This chapter reviews recent findings that reveal the importance of RNA splicing and processing rearrangements in the expression and regulation of immunoglobulin heavy chain genes. RNA rearrangements greatly increase the versatility of immunoglobulin heavy chain genes by generating alternate forms of mRNA, with different functions, from one transcription unit. Both secreted and membrane mRNA species for all heavy chain classes are generated by alternative RNA splicing pathways. The immunoglobulin molecule can exist in two very different environments: (1) in the lymphocyte cell membrane as a surface antigen receptor and (2) in the circulation as a secreted antibody. Most secreted immunoglobulin molecules consist of two identical light chains and two identical heavy chains. Each chain is composed of a series of homologous domains, the NH2-terminal one being the variable (V) region and the remainder forming the constant (C) region. Complex transcription units and posttranscriptional control of chromosomal genes are not unique to immunoglobulin heavy chain genes. The findings collected in the chapter clearly establish the importance of posttranscriptional RNA processing mechanisms in regulation of genes in eukaryotic cells.

Expression of the human insulin gene and cDNA in a heterologous mammalian system.

The human insulin gene or the corresponding cDNA has been inserted into the early region of a simian virus 40 vector in which all SV40 splice junctions were deleted while the early promoter and polyadenylation regions remained intact. The expression of insulin-coding sequences was tested in permissive monkey COS cells. The insulin cDNA was transcribed from the early promoter to produce a stable polyadenylated RNA which was translated, and immunoreactive human proinsulin accumulated in the medium. Thus RNA splicing is not obligatory for insulin expression in this system. The genomic insulin transcript was also initiated from the SV40 promoter and terminated at the SV40 polyadenylation site. S1 endonuclease mapping revealed that the transcript is processed via two alternative splicing pathways within the insulin gene. About one-third of the total transcripts are processed normally with removal of the two insulin-specific introns. This transcript is apparently translated normally since immunoreactive proinsulin accumulates in the medium. About two-thirds of the transcripts are processed via an alternative splicing pathway involving a new splice acceptor site located within the coding region of the insulin gene. This results in a codon frameshift such that translation would produce a novel chimeric peptide containing the insulin NH2-terminal B chain, but a different COOH terminus containing human and SV40 sequences. A peptide of the predicted size is detected in the COS cell extract.

Alternative splicing pathways exist in the formation of adenoviral late messenger RNAs

Total rapidly labeled RNA was extracted from nuclei of HeLa cells late after infection with adenovirus type 2. Most of this nuclear RNA is transcribed from the major late transcription unit (16·2 to 100·0 map units (m.u.)). To study the cleavage reactions that are involved in RNA splicing, we used the S 1 nuclease mapping technique with HinIII B (16·8 to 31·5 m.u.) and XhoI F (15·5 to 22·4 m.u.) restriction fragments as DNA probes. The S 1 mapping data showed that both intron 1 (16·3 to 19·1 m.u.) and intron 2 (19·4 to 26·2 m.u.) can be excised in more than one step. Kinetic labeling and pulse-chase experiments demonstrated that certain cleavage sites in the RNA are used within three minutes after the start of transcription, while other cleavages occur only after a significant time-lag. The use of 5,6-dichloro-1-β- d-ribofuranosylbenzimidazole enabled us to label the nuclear RNA exclusively from the 5′ end. Such an approach showed that the first cleavages are introduced in the nascent RNA before the RNA polymerase has passed more than 2000 nucleotides beyond the cleavage site. The chronology of the appearance of processing intermediates that results from cleavage of the RNA indicates that preferentially intron 1 is removed before intron 2. This is in agreement with the finding that leader 1 is ligated to leader 2 before ligation of leader 2 to leader 3 takes place. However, we have found that alternative splicing pathways exist in the excision of introns 1 and 2, demonstrating that cleavage in intron 2 may occur before intron 1 is attacked by the splicing enzymes.

Human growth hormone DNA sequence and mRNA structure: possible alternative splicing.

We have determined the complete sequence of the human growth hormone (hGH) gene and the position of the mature 5' end of the hGH mRNA within the sequence. Comparison of this sequence with that of a cloned hGH cDNA shows that the gene is interrupted by four intervening sequences. S1 mapping shows that one of these intervening sequences has two different 3' splice sites. These alternate splicing pathways generate hGH peptides of different sizes which are found in normal pituitaries. Comparison of sequences near the 5' end of the hGH mRNA with a similar region of the alpha subunit of the human glycoprotein hormones reveals an unexpected region of homology between these otherwise unrelated peptide hormones.