We have investigated the composition of the earliest detectable complex (H) assembled on pre-mRNA during the in vitro splicing reaction. We show that most of the proteins in this complex correspond to heterogeneous nuclear ribonucleoproteins (hnRNP), a set of abundant RNA-binding proteins that bind nascent RNA polymerase II transcripts in vivo. Thus, these studies establish a direct parallel between the initial events of RNA processing in vitro and in vivo. In contrast to previous studies, in which total hnRNP particles were isolated from mammalian nuclei, we determined the hnRNP composition of complexes assembled on individual RNAs of defined sequence. We found that a unique combination of hnRNP proteins is associated with each RNA. Thus, our data provide direct evidence for transcript-dependent assembly of pre-mRNA in hnRNP complexes. The observation that pre-mRNA is differentially bound by hnRNP proteins prior to spliceosome assembly suggests the possibility that RNA packaging could play a central role in the mechanism of splice site selection, as well as other posttranscriptional events.During transcription, nascent heterogeneous nuclear RNAs (hnRNAs) associate with a distinct set of abundant nuclear proteins, known as heterogeneous nuclear ribonucleoproteins (hnRNP) to form hnRNP complexes (see references 10, 12, and 14 for reviews). The major hnRNP proteins detected by sodium dodecyl sulfate (SDS)-gel electrophoresis of isolated mammalian hnRNP complexes (also referred to as hnRNP particles) include a group of 35-to 45-kDa proteins and groups of 68-and 120-kDa proteins (7,12). However, at least 20 distinct hnRNP proteins have been identified by two-dimensional gel electrophoresis (36). On the basis of the observations that hnRNP proteins bind to nascent pre-mRNA and that splicing can be visualized on these transcripts in the electron microscope (2, 32), hnRNP complexes are thought to be the substrate for posttranscriptional processing events (12).In vitro studies have shown that pre-mRNA splicing takes place within spliceosomes, large multicomponent complexes containing Ul, U2, U4, U5, and U6 small nuclear RNPs (snRNPs) (see references 21 and 28 for reviews) and a number of non-snRNP proteins (26,27,38). Spliceosome assembly occurs by means of a stepwise pathway. PremRNA is first assembled into an ATP-independent complex which lacks snRNPs (15,19,24,38). This complex (H) does not appear to be specific to splicing because it assembles as efficiently on natural pre-mRNAs as on RNAs lacking functional splice sites (19,24,38). However, H complex assembly precedes that of any splicing-specific complexes (30), indicating that this complex is either a spliceosome precursor or that it disassembles prior to spliceosome formation.Following H complex assembly, the first complex specific to splicing, E (early) complex, is an ATP-independent com-

Heterogeneous nuclear ribonucleoprotein (hnRNP) complexes, the structures that contain heterogeneous nuclear RNA and its associated proteins, constitute one of the most abundant components of the eukaryotic nucleus. hnRNPs appear to play important roles in the processing, and possibly also in the transport, of mRNA. hnRNP C proteins (Cl, Mr of 41,000; C2, Mr of 43,000 [by sodium dodecyl sulfate-polyacrylamide gel electrophoresis]) are among the most abundant pre-mRNA-binding proteins, and they bind tenaciously to sequences relevant to pre-mRNA processing, including the polypyrimidine stretch of introns (when it is uridine rich). C proteins are found in the nucleus during the interphase, but during mitosis they disperse throughout the cell. They have been shown previously to be phosphorylated in vivo, and they can be phosphorylated in vitro by a casein kinase type II. We have identified and partially purified at least two additional C protein kinases. One of these, termed Cs kinase, caused a distinct mobility shift of C proteins on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These phosphorylated C proteins, the Cs proteins, were the prevalent forms of C proteins during mitosis, and Cs kinase activity was also increased in extracts prepared from mitotic cells. Thus, hnRNP C proteins undergo cell cycle-dependent phosphorylation by a cell cycle-regulated protein kinase. Cs kinase activity appears to be distinct from the well-characterized mitosis-specific histone Hi kinase activity. Several additional hnRNP proteins are also phosphorylated during mitosis and are thus also potential substrates for Cs kinase. These novel phosphorylations may be important in regulating the assembly and disassembly of hnRNP complexes and in the function or cellular localization of RNA-binding proteins.RNA polymerase II transcripts, collectively termed heterogeneous nuclear RNAs (hnRNAs), associate as they are transcribed in the nuclei of eukaryotic cells with a specific subset of proteins to form heterogeneous nuclear ribonucleoprotein (hnRNP) complexes (12, 15). In human HeLa cells, these complexes contain, in addition to hnRNA, an assortment of at least 20 different abundant polypeptides (7, 30). As this association persists throughout their nuclear residency, it is assumed that the ensuing events that pre-mRNAs undergo to become functional translatable cytoplasmic mRNAs occur on hnRNPs rather than on naked RNA molecules.Of the ca. 20 major hnRNP proteins identified, the C proteins have occupied much of the focus of hnRNP research for several reasons. They are a prominent doublet (Cl [41 kDa] and C2 [43 kDa]) in HeLa cells) of nuclear acidic proteins which remain bound to the RNA at salt concentrations at which most of the other hnRNPs dissociate (3, 7). C proteins can also be readily UV-cross-linked to poly(A)-containing nuclear RNA in vivo (13,14,42). Immunofluorescence microscopy with monoclonal antibodies in a variety of vertebrate cells has shown that C proteins are localized to the nucleoplasm and are excluded fro...