Myotonic dystrophy (DM) is an autosomal dominant neuromuscular disease that is associated with a (CTG)n repeat expansion in the 3'-untranslated region of the myotonin protein kinase (Mt-PK) gene. This study reports the isolation and characterization of a (CUG)n triplet repeat pre-mRNA/mRNA binding protein that may play an important role in DM pathogenesis. Two HeLa cell proteins, CUG-BP1 and CUG-BP2, have been purified based upon their ability to bind specifically to (CUG)8 oligonucleotides in vitro. While CUG-BP1 is the major (CUG)8-binding activity in normal cells, nuclear CUG-BP2 binding activity increases in DM cells. Both CUG-BP1 and CUG-BP2 have been identified as isoforms of a novel heterogeneous nuclear ribonucleoprotein (hnRNP), hNab50. The CUG-BP/hNab50 protein is localized predominantly in the nucleus and is associated with polyadenylated RNAs in vivo. In vitro RNA-binding/photocrosslinking studies demonstrate that CUG-BP/hNab50 binds to RNAs containing the Mt-PK 3'-UTR. We propose that the (CUG)n repeat region in Mt-PK mRNA is a binding site for CUG-BP/hNab50 in vivo, and triplet repeat expansion leads to sequestration of this hnRNP on mutant Mt-PK transcripts.

A variety of nuclear ribonucleoproteins are believed to associate directly with nascent RNA polymerase II transcripts and remain associated during subsequent nuclear RNA processing reactions, including pre-mRNA polyadenylation and splicing as well as nucleocytoplasmic mRNA transport. To investigate the functions of these proteins by using a combined biochemical and genetic approach, we have isolated nuclear polyadenylated RNA-binding (NAB) proteins from Saccharomyces cerevisiae. Living yeast cells were irradiated with UV light to covalently cross-link proteins intimately associated with RNA in vivo. Polyadenylated RNAs were then selectively purified, and the covalent RNA-protein complexes were used to elicit antibodies in mice. Both monoclonal and polyclonal antibodies which detect a variety of NAB proteins were prepared. Here we characterize one of these proteins, NAB2. NAB2 is one of the major proteins associated with nuclear polyadenylated RNA in vivo, as detected by UV light-induced cross-linking. Cellular immunofluorescence, using both monoclonal and polyclonal antibodies, demonstrates that the NAB2 protein is localized within the nucleus. The deduced primary structure of NAB2 indicates that it is composed of at least two distinct types of RNA-binding motifs: (i) an RGG box recently described in a variety of heterogeneous nuclear RNA-, pre-rRNA-, mRNA-, and small nucleolar RNA-binding proteins and (ii) CCCH motif repeats related to the zinc-binding motifs of the largest subunit of RNA polymerases I, II, and III. In vitro RNA homopolymer/ single-stranded DNA binding studies indicate that although both the RGG box and CCCH motifs bind poly(G), poly(U), and single-stranded DNA, the CCCH motifs also bind to poly(A). NAB2 is located on chromosome VII within a cluster of ribonucleoprotein genes, and its expression is essential for cell growth.Gene expression is regulated at multiple levels following the initiation of transcription by RNA polymerase II. Nascent pre-mRNA transcripts are extensively modified to generate mRNAs (59). These modifications, all of which occur in the nucleus, may include 5'-end capping (46, 52), 3'-end cleavage and polyadenylation (64), splicing (27, 53), and modification of individual bases (16). All of these activities occur while pre-mRNAs are associated with a set of nuclear factors which may include both heterogeneous nuclear ribonucleoproteins (hnRNPs) and small nuclear ribonucleoproteins (snRNPs) (21,23,30,61). Once formed in the nucleus, mRNAs are transported into the cytoplasm by an unknown mechanism (40). mRNA turnover in both the nucleus and cytoplasm may also dramatically influence the amount of each type of mRNA available for translation (18).Although considerable information exists concerning the functional role of snRNPs in nuclear RNA processing (27, 30, 53, 61), very little is known about the specific functions of individual hnRNPs. Previous work focused on the idea that hnRNPs are primarily pre-mRNA-packaging proteins functionally analogous to the role that histones play...

Abstract. To study the functions of heterogeneous nuclear ribonucleoproteins (hnRNPs), we have characterized nuclear polyadenylated RNA-binding (Nab) proteins from Saccharomyces cerevisiae. Nablp, Nab2p, and Nab3p were isolated by a method which uses UV light to cross-link proteins directly bound to poly(A) + RNA in vivo. We have previously characterized Nab2p, and demonstrated that it is structurally related to human hnRNPs. Here we report that Nablp is identical to the Npl3p/Nop3p protein recently implicated in both nucleocytoplasmic protein shuttling and prerRNA processing, and characterize a new nuclear polyadenylated RNA-binding protein, Nab3p. The intranuclear distributions of the Nab proteins were analyzed by three-dimensional immunofluorescence optical microscopy. All three Nab proteins are predominantly localized within the nucleoplasm in a pattern similar to the distribution of hnRNPs in human cells. The NAB3 gene is essential for cell viability and encodes an acidic ribonucleoprotein. Loss of Nab3p by growth of a GAL::nab3 mutant strain in glucose results in a decrease in the amount of mature ACT1, CYH2, and TP//mRNAs, a concomitant accumulation of unspliced ACTI pre-mRNA, and an increase in the ratio of unspliced CYH2 pre-mRNA to mRNA. These results suggest that the Nab proteins may be required for packaging pre-mRNAs into ribonucleoprotein structures amenable to efficient nuclear RNA processing. HETEROGENEOUS nuclear RNAs (hnRNAs) t are the products of RNA polymerase II transcription, and include polyadenylated and nonpolyadenylated premRNAs and mRNAs as well as several uncharacterized RNAs (Dreyfuss et al., 1993). An array of nuclear factors bind to hnRNAs during transcription, including heterogeneous nuclear ribonucleoproteins (hnRNPs) and small nuclear RNP (snRNP) particles (Amero et al., 1992;Matunis et al., 1993). Numerous studies have established the roles of snRNP particles in a variety of nuclear processes such as pre-mRNA splicing (Green, 1991;Guthrie, 1991; Ruby and Abelson, 1991;Baserga and Steitz, 1993). In contrast, the functions of hnRNPs have remained unclear. HnRNPs are nuclear RNA-binding proteins whose primary and stable RNA-binding site is hnRNA. Current ideas about the functional roles of hnRNPs in pre-mRNA processing have Address all correspondence to M.