The MEF2 site is an essential element of muscle enhancers and promoters that is bound by a nuclear activity found, so far, only in muscle and required for tissue-specific transcription. We have cloned a group of transcription factors from human muscle that are responsible for this activity: They are present in muscle-specific DNA-binding complexes, have a target sequence specificity identical to that of the endogenous activity, and are MEF2 site-dependent transcriptional activators. These MEF2 proteins comprise several alternatively spliced isoforms from one gene and a related factor encoded by a second gene. All share a conserved amino-terminal DNA-binding domain that includes the MADS homology. MEF2 transcripts are ubiquitous but accumulate preferentially in skeletal muscle, heart, and brain. Specific alternatively spliced isoforms are restricted to these tissues, correlating exactly with the presence of endogenous MEF2 activity. Furthermore, MEF2 protein is detected only in skeletal and cardiac muscle nuclei and not in myoblast and nonmuscle cells. Thus, post-transcriptional regulation is important in the generation of tissue-specific MEF2 activity. Cardiac and smooth, as well as skeletal, muscles contain functionally saturating levels of MEF2 trans-activating factors that are absent in nonmuscle cells. Moreover, MEF2 is induced in nonmuscle cells by MyoD; however, MEF2 alone is insufficient to produce the full muscle phenotype. Implications for the molecular mechanisms of myogenesis are considered.

Previously, we characterized cDNAs encoding polypyrimidine tract-binding protein {PTB) and showed that a complex between PTB and a 100-kD protein was necessary for pre-mRNA splicing. In this paper we have used two different in vitro-binding assays to confirm and extend the interaction between these two proteins. Peptide sequence information was used to clone and sequence cDNAs encoding alternatively spliced forms of the 100-kD protein. It contains two consensus RNA-binding domains and an unusual amino terminus rich in proline and glutamine residues. The protein is highly basic and migrates anomalously on SDS gels. Owing to its interaction with PTB and its role in pre-mRNA splicing, we have termed the 100-kD protein PTB-associated splicing factor (PSF). The RNA-binding properties of PSF are apparently identical to those of PTB. Both proteins, together and independently, bind the polypyrimidine tract of mammalian introns. Biochemical complementation, antibody inhibition, and immunodepletion experiments demonstrate that PSF is an essential pre-mRNA splicing factor required early in spliceosome formation. Bacterially synthesized PSF is able to complement immunodepleted extracts and restore splicing activity. Despite association with PSF, complementary experiments with antibodies against PTB do not suggest an essential role for PTB in pre-mRNA splicing.[Key Words: Pre-mRNA splicing; polypyrimidine tract-binding protein; PTB-associated splicing factor; cloning] Received December 9, 1992; revised version accepted January 5, 1993.The removal of intervening sequences from pre-mRNAs requires the formation of splicing complexes in an ordered, ATP-dependent pathway (for reviews, see Sharp 1987; Krainer and Maniatis 1988;Steitz et al. 1988;Smith et al. 1989a;Green 1991;Guthrie 1991). The assembly of multiple factors into these spliceosomes leads to intron excision through a two-step cleavage-ligation reaction. The development of mammalian in vitro splicing systems has allowed the identification of several cisand trans-acting components necessary for pre-mRNA splicing. Chief among the required cis-acting elements are a consensus sequence at the 5' splice site, a branchpoint sequence and adjacent polypyrimidine tract, and the 3' splice site AG dinucleotide (Mount 1982;Ohshima and Gotoh 1987). Among the best characterized of the trans-acting factors are the family of small nuclear ribonucleoprotein particles, the U snRNPs (Steitz et al. 1988). U1 and U2 snRNP initially base-pair with the 5' splice site and the branchpoint sequence, respectively, followed by the joining of U4/U6 and U5 as part of a preformed triple snRNP (Behrens and Lfihrmann 1991). Before catalysis, multiple base-pairing interactions between the pre-mRNA and small nuclear RNAs (snRNAs) Steitz 1992), multiple protein components, both snRNP and non-snRNP, are also required for pre-mRNA splicing. Each snRNP particle consists of one or two small RNAs complexed with common and unique proteins (Liihrmann 1988). Additional snRNPs are found associated with multi-snRN...

Expression of the cardiac myosin isozymes is regulated during development, by hormonal stimuli and hemodynamic load. In this study, the levels of expression of the two isoforms (a and ,j) of myosin heavy chain (MHC) during cardiac hypertrophy were investigated at the messenger RNA (mRNA) and protein levels. In normal control and sham-operated rats, the a-MHC mRNA predominated in the ventricular myocardium. In response to aortic coarctation, there was a rapid induction of the #-MHC mRNA followed by the appearance of comparable levels of the jN-MHC protein in parallel to an increase in the left ventricular weight. Administration of thyroxine to coarctated animals caused a rapid deinduction of 0-MHC and induction of a-MHC, both at the mRNA and protein levels, despite progression of left ventricular hypertrophy. These results suggest that the MHC isozyme transition during hemodynamic overload is mainly regulated by pretranslational mechanisms, and that a complex interplay exists between hemodynamic and hormonal stimuli in MHC gene expression.

In mammalian intron splicing, the mechanism by which the 3' splice site AG is accurately and efficiently identified has remained unresolved. We have previously proposed that the 3' splice site in mammalian introns is located by a scanning mechanism for the first AG downstream of the branch point-polypyrimidine tract. We now present experiments that lend further support to this model while identifying conditions under which competition can occur between adjacent AGs. The data show that the 3' splice site is identified as the first AG downstream from the branch point by a mechanism that has all the characteristics expected of a 5'-to-3' scanning process that starts from the branch point rather than the pyrimidine tract. Failure to recognize the proximal AG may arise, however, from extreme proximity to the branch point or sequestration within a hairpin. Once an AG has been encountered, the spliceosome can still see a limited stretch of downstream RNA within which an AG more competitive than the proximal one may be selected. Proximity to the branch point is a major determinant of competition, although steric effects render an AG less competitive in close proximity (-12 nucleotides). In addition, the nucleotide preceding the AG has a striking influence upon competition between closely spaced AGs. The order of competitiveness, CAG = UAG > AAG > GAG, is similar to the nucleotide preference at this position in wild-type 3' splice sites. Thus, 3' splice site selection displays properties of both a scanning process and competition between AGs based on immediate sequence context. This refined scanning model, incorporating elements of competition, is the simplest interpretation that is consistent with all of the available data.Pre-mRNA splicing is a mandatory consequence of the split nature of eukaryotic genes. The production of functional mRNA from primary transcripts requires the accurate removal of the noncoding introns and ligation of adjacent exons. Pre-mRNA splicing provides an important level at which gene expression can be regulated; pairing of different combinations of splice sites leads to the production of tissue-specific mRNAs that code for distinct protein isoforms or that switch off gene expression via the premature termination of open reading frames (52). The splicing pathway proceeds via a well-characterized two-step reaction of successive transesterifications (14,30,39). The integrity of open reading frames demands that the sites of these reactions be specified to a high degree. This specificity is achieved by the presence of consensus sequences at the 5' splice site ([A/C]AG GURAGU) and three elements associated with the 3' end of the intron, the branch point (YNYURAY), the polypyrimidine tract downstream of the branch point, and the 3' splice site (YAGIG) ( (46,48,60,63), while U2 base pairs with the branch point sequence (31,60,61,64). In yeast cells, U5 RNA has been shown to interact with exon sequences adjacent to both splice sites (28) and Ul has recently been shown to base pair with the 3' splice si...

We have investigated the developmental transitions of myosin heavy chain (MHC) gene expression in the rat extraocular musculature (EOM) at the mRNA level using Sl-nuclease mapping techniques and at the protein level by polypeptide mapping and immunochemistry. We have isolated a genomic clone, designated 2,10B3, corresponding to an MHC gene which is expressed in the EOM fibers (recti and oblique muscles) of the adult rat but not in hind limb muscles. Using cDNA and genomic probes for MHC genes expressed in skeletal (embryonic, neonatal, fast oxidative, fast glycolytic, and slow/cardiac ~-MHC), cardiac (e-MHC), and EOM (X 10B3) muscles, we demonstrate the concomitant expression at the mRNA level of at least six different MHC genes in adult EOM. Protein and immunochemical analyses confirm the presence of at least four different MHC types in EOM. Immunocytochemistry demonstrates that different myosin isozymes tend to segregate into individual myofibers, although some fibers seem to contain more than one MHC type.The results also show that the EOM fibers exhibit multiple patterns of MHC gene regulation. One set of fibers undergoes a sequence of isoform transitions similar to the one described for limb skeletal muscles, whereas other EOM myofiber populations arrest the MHC transition at the embryonic, neonatal/adult, or adult EOM-specific stage. Thus, the MHC gene family is not under the control of a strict developmental clock, but the individual genes can modify their expression by tissue-specific and/or environmental factors.