hnRNP A1 is a pre‐mRNA binding protein that antagonizes the alternative splicing activity of splicing factors SF2/ASF or SC35, causing activation of distal 5′ splice sites. The structural requirements for hnRNP A1 function were determined by mutagenesis of recombinant human hnRNP A1. Two conserved Phe residues in the RNP‐1 submotif of each of two RNA recognition motifs appear to be involved in specific RNA‐protein interactions and are essential for modulating alternative splicing. These residues are not required for general pre‐mRNA binding or RNA annealing activity. The C‐terminal Gly‐rich domain is necessary for alternative splicing activity, for stable RNA binding and for optimal RNA annealing activity. hnRNP A1B, which is an alternatively spliced isoform of hnRNP A1 with a longer Gly‐rich domain, binds more strongly to pre‐mRNA but has only limited alternative splicing activity. In contrast, hnRNP A2 and B1, which have 68% amino acid identity with hnRNP A1, bind more weakly to pre‐mRNA and have stronger splice site switching activities than hnRNP A1. We propose that specific combinations of antagonistic hnRNP A/B and SR proteins are involved in regulating alternative splicing of distinct subsets of cellular premRNAs.

The first component known to recognize and discriminate among potential 5 splice sites (5SSs) in pre-mRNA is the U1 snRNP. However, the relative levels of U1 snRNP binding to alternative 5SSs do not necessarily determine the splicing outcome. Strikingly, SF2/ASF, one of the essential SR protein-splicing factors, causes a dose-dependent shift in splicing to a downstream (intron-proximal) site, and yet it increases U1 snRNP binding at upstream and downstream sites simultaneously. We show here that hnRNP A1, which shifts splicing towards an upstream 5SS, causes reduced U1 snRNP binding at both sites. Nonetheless, the importance of U1 snRNP binding is shown by proportionality between the level of U1 snRNP binding to the downstream site and its use in splicing. With purified components, hnRNP A1 reduces U1 snRNP binding to 5SSs by binding cooperatively and indiscriminately to the pre-mRNA. Mutations in hnRNP A1 and SF2/ASF show that the opposite effects of the proteins on 5SS choice are correlated with their effects on U1 snRNP binding. Cross-linking experiments show that SF2/ASF and hnRNP A1 compete to bind pre-mRNA, and we conclude that this competition is the basis of their functional antagonism; SF2/ASF enhances U1 snRNP binding at all 5SSs, the rise in simultaneous occupancy causing a shift in splicing towards the downstream site, whereas hnRNP A1 interferes with U1 snRNP binding such that 5SS occupancy is lower and the affinities of U1 snRNP for the individual sites determine the site of splicing.Alternative splicing of pre-mRNA is responsible for the production of multiple mRNA and protein products from individual genes. In many cases, different protein isoforms have unique functions, and their production is tightly regulated at the splicing level. Although a common form of alternative splicing involves the omission or skipping of specific exons during splicing, there are many examples of alternative splicing in which two or more alternative 5Ј splice sites (5ЈSSs) compete for joining to a single 3Ј splice site. In such cases, both sites may be used ubiquitously, or their use may be stringently regulated. Similarly, the deliberate introduction of a duplicate 5ЈSS may result in use of either site or both sites, depending on the precise context, including the sequence of the sites, their relative 5Ј-3Ј order, separation, adjacent sequences, or secondary structures (9,28,31,33,46,57,58,66,73). Some of these influences reflect the activity of trans-acting factors.One trans-acting factor is the U1 snRNP, the RNA component of which forms base pairs across the 5ЈSSs. The strength of base pairing correlates with the choice of 5ЈSSs in vivo, at least in some circumstances (39,77,95), suggesting that a low probability of binding by U1 snRNP and different affinities for various 5ЈSSs can dictate 5ЈSS preferences. This idea is consistent with the observation in Saccharomyces cerevisiae that interactions by components of the U1 snRNP with the capbinding complex or adjacent pre-mRNA sequences can influence 5ЈSS selection (3...

Thyroid hormone (T 3 ) coordinates growth, differentiation, and metabolism by binding to nuclear thyroid hormone receptors (TRs). The TR␣ gene encodes T 3 -activated TR␣1 (NR1A1a) as well as an antagonistic, non-T 3 -binding alternatively spliced product, TR␣2 (NR1A1b). Thus, the TR␣1/TR␣2 ratio is a critical determinant of T 3 action. However, the mechanisms underlying this post-transcriptional regulation are unknown. We have identified a non-consensus, TR␣2-specific 5 splice site and conserved intronic sequences as key determinants of TR␣ mRNA processing. In addition to these cis-acting elements, a novel regulatory feature is the orphan receptor RevErbA␣ (NR1D1) gene, which is transcribed from the opposite direction at the same locus and overlaps the TR␣2 coding region. RevErbA␣ gene expression correlates with a high TR␣1/TR␣2 ratio in a number of tissues. Here we demonstrate that coexpression of RevErbA␣ and TR␣ regulates the TR␣1/TR␣2 ratio in intact cells. Thus, both cis-and trans-regulatory mechanisms contribute to cell-specific post-transcriptional regulation of TR gene expression and T 3 action. Thyroid hormone receptors (TRs)1 mediate the diverse physiological effects of thyroid hormone (T 3 ) (1-3). TRs are encoded by two closely related genes, erbA␣ and erbA␤, in all vertebrates (4). Additional receptor diversity is generated by alternative processing of the TR␣ and TR␤ pre-mRNAs. The alternatively spliced isoforms of TR␣, TR␣1 (NR1A1a) (5) and TR␣2 (NR1A1b), are of particular interest. Although both are widely expressed, they are functionally antagonistic. Due to its variant C terminus, TR␣2, unlike TR␣1, does not bind T 3 and lacks the major activation function present in other TRs (6, 7). The dominant negative activity of TR␣2 appears to reflect both competition for binding to TR target genes as well as altered protein-protein interactions (8 -11).Expression of the two TR␣ isoforms is highly regulated, with each mRNA expressed in a tissue-specific and developmentally regulated fashion. In some tissues TR␣2 represents a relatively minor fraction of the TR-related isoforms, while in other tissues such as brain, kidney, and testes, TR␣2 is the most abundant isoform (12, 13). A developmental increase in the T 3 responsiveness of some cells correlates with a decrease in the relative expression of TR␣2 mRNA, suggesting that TR␣2 expression modulates T 3 action (13). Thus, TR␣2 may act as a tissuespecific antagonist of T 3 -responsive gene activation.Interestingly, the alternative post-transcriptional processing of the TR␣ transcript that gives rise to TR␣2 mRNA occurs exclusively in mammals (14 -17). The mammalian TR␣ gene is also remarkable in that it partially overlaps the gene for another nuclear receptor that is encoded on the opposite DNA strand. This receptor gene, RevErbA␣ (RevErb, NR1D1), is convergently transcribed with respect to the TR␣ gene such that its 3Ј end overlaps sequences coding for TR␣2, but not TR␣1 (18,19). The unusual organization of these two genes, which code for structurally re...

Within the nucleus, pre-mRNA molecules are complexed with a set of proteins to form heterogeneous nuclear ribonucleoprotein complexes. Al, an abundant RNA binding protein present in these complexes, has been shown to bind selectively to single-stranded RNAs and destabilize basepairing interactions. In this study Al is shown to promote the rate of annealing of complementary RNA strands >300-fold under a wide range of salt concentration and temperature. Maximal annealing is observed under saturating or near saturating concentrations of protein, but annealing decreases sharply at both higher and lower concentrations of Al. Kinetic analysis shows that the rate of annealing is not strictly rst or second order with respect to RNA at a ratio of protein/RNA that gives optimal rates of annealing. This result suggests that Al protein may affect more than one step in the annealing reaction. Two polypeptides representing different domains of Al were also examined for annealing activity. UPl, a proteolytic fragment that represents the N-terminal two-thirds of Al, displays very limited annealing activity. In contrast, a peptide consisting of 48 amino acid residues from the glycine-rich C-terminal region promotes annealing at a rate almost onequarter that observed with intact Al. The RNARNA annealing activity of Al may play a role in pre-mRNA splicing and other aspects of nuclear mRNA metabolism.RNARNA base pairing plays a key role in regulating the transfer and expression ofgenetic information at many levels, including replication, transcription, mRNA processing, and translation (1). Although base pairing of complementary RNAs is sometimes regarded as a spontaneous process, in vitro annealing of nucleic acids of the size and complexity of naturally occurring RNA molecules is slow under conditions of ionic strength and temperature approximating those inside most cells (2, 3). In vivo, base-pairing interactions may be mediated by proteins that promote or destabilize formation of double-stranded structure.Recently, several types of proteins have been characterized that unwind or destabilize double-stranded RNA. These include helicases (4, 5), enzymes that deaminate adenosine residues in double-stranded RNA (6, 7), and single-strandspecific RNA binding proteins (8). The function of most such proteins is poorly understood, although helicases are known to be required for the splicing and translation of mRNA (4, 9). Other proteins accelerate the rate of RNARNA annealing. These include the Rom (or Rop) protein, which regulates plasmid replication (10), and a small RNA-binding protein found in retroviral cores (11). This laboratory has shown (12) that RNARNA annealing proteins are abundant in nuclear extracts active in pre-mRNA splicing. In this report we investigate the ability of an abundant nuclear protein, the heterogeneous nuclear ribonucleoprotein (hnRNP) Al (13,14), to accelerate the rate of RNA-RNA annealing under a wide range of conditions.Previous studies have shown that Al, like a number of other DNA and RNA binding...

The erbAalpha gene encodes two alpha-thyroid hormone receptor isoforms, TRalpha1 and TRalpha2, which arise from alternatively processed mRNAs, erbAalpha1 (alpha1) and erb alpha2 (alpha2). The splicing and alternative polyadenylation patterns of these mRNAs resemble that of mRNAs encoding different forms of immunoglobulin heavy chains, which are regulated at the level of alternative processing during B cell differentiation. This study examines the levels of erbAalpha mRNA in eight B cell lines representing four stages of differentiation in order to determine whether regulation of the alternatively processed alpha1 and alpha2 mRNAs parallels the processing of immunoglobulin heavy chain mRNAs. Results show that the pattern of alpha1 and alpha2 mRNA expression is clearly different from that observed for immunoglobulin heavy chain mRNAs. B cell lines display characteristic ratios of alpha1/alpha2 mRNA at distinct stages of differentiation. Furthermore, expression of an overlapping gene, Rev-ErbAalpha (RevErb), was found to correlate strongly with an increase in the ratio of alpha1/alpha2 mRNA. These results suggest that alternative processing of erbAalpha mRNAs is regulated by a mechanism which is distinct from that regulating immunoglobulin mRNA. The correlation between RevErb and erbAalpha mRNA is consistent with negative regulation of alpha2 via antisense interactions with the complementary RevErb mRNA.