proteins are the major protein components of heterogeneous ribonucleoprotein particles (hnRNPs), which contain heterogeneous nuclear RNA (hnRNA), a collective term referring to pre-mRNA and other nuclear RNAs of various sizes. The proteins in these ribonucleoprotein particles can be dramatically visualized with fluorescent-labeled monoclonal antibodies (see the chapter opening figure).

Researchers identified hnRNP proteins by first exposing cultured cells to high-dose UV irradiation, which causes cova-lent cross-links to form between RNA bases and closely associated proteins. Chromatography of nuclear extracts from treated cells on an oligo-dT cellulose column, which binds RNAs with a poly(A) tail, was used to recover the proteins that had become cross-linked to nuclear polyadenylated RNA. Subsequent treatment of cell extracts from unirradiated cells with monoclonal antibodies specific for the major proteins identified by this cross-linking technique revealed a complex set of abundant hnRNP proteins ranging in size from 34 to 120 kDa.

Like transcription factors, most hnRNP proteins have a modular structure. They contain one or more RNA-binding domains and at least one other domain that is thought to interact with other proteins. Several different RNA-binding motifs have been identified by constructing deletions of hnRNP proteins and testing their ability to bind RNA.

Conserved RNA-Binding Motifs The RNA recognition motif (RRM), also called the RNP motif and the RNA-

▲ FIGURE 12-3 Structure of the RRM domain and its interaction with RNA. (a) Diagram of the RRM domain showing the two a helices and four p strands that characterize this motif. The conserved RNP1 and RNP2 regions are located in the two central p strands. (b) Surface representation of the two RRM domains in Drosophila Sex-lethal (Sxl) protein, which binds a nine-base sequence in transformer pre-mRNA (green). The two RRMs are oriented like the two parts of an open pair of castanets, with binding domain (RBD), is the most common RNA-binding domain in hnRNP proteins. This «80-residue domain, which occurs in many other RNA-binding proteins, contains two highly conserved sequences (RNP1 and RNP2) that allow the motif to be recognized in newly sequenced genes. X-ray crys-tallographic analysis has shown that the RRM domain consists of a four-stranded p sheet flanked on one side by two a helices. The conserved RNP1 and RNP2 sequences lie side by side on the two central p strands, and their side chains make multiple contacts with a single-stranded region of RNA (Figure 12-3). The single-stranded RNA lies across the surface of the p sheet, with the central RNP1 and RNP2 strands forming a positively charged surface that interacts with the negatively charged RNA phosphates.

The RGG box, another RNA-binding motif found in hnRNP proteins, contains five Arg-Gly-Gly (RGG) repeats with several interspersed aromatic amino acids. Although the structure of this motif has not yet been determined, its argininerich nature is similar to the RNA-binding domains of the HIV Tat protein. The 45-residue KH motif is found in the hnRNP K protein and several other RNA-binding proteins; commonly two or more copies of the KH motif are interspersed with RGG repeats. The three-dimensional structure of representative KH domains is similar to that of the RRM domain but smaller, consisting of a three-stranded p sheet supported from one side by two a helices. RNA binds to the KH motif by interacting with a hydrophobic surface formed by the a helices and one the p sheet of RRM1 facing upward and the p sheet of RRM2 facing downward. Positively charged regions in Sxl protein are shown in shades of blue; negatively charged regions, in shades of red. The pre-mRNA is bound to the surfaces of the positively charged p sheets, making most of its contacts with the RNP1 and RNP2 regions of each RRM. [Part (a) adapted from K. Nagai et al., 1995, Trends Biochem. Sci. 20: 235; part (b) after N. Harada et al., 1999, Nature 398:579.]

^-strand. Thus, despite the similarity in their structures, RRM and KH domains interact differently with RNA.

Functions of hnRNP Proteins The association of pre-mRNAs with hnRNP proteins prevents formation of short secondary structures dependent on base-pairing of complementary regions, thereby making the pre-mRNAs accessible for interaction with other RNA molecules or proteins. Pre-mRNAs associated with hnRNP proteins present a more uniform substrate for further processing steps than would free, unbound pre-mRNAs, each type of which forms a unique secondary structure dependent on its specific sequence.

Binding studies with purified hnRNP proteins suggest that different hnRNP proteins associate with different regions of a newly made pre-mRNA molecule. For example, the hnRNP proteins A1, C, and D bind preferentially to the pyrimidine-rich sequences at the 3' ends of introns (see later discussion). This observation suggests that some hnRNP proteins may interact with the RNA sequences that specify RNA splicing or cleavage/polyadenylation and contribute to the structure recognized by RNA-processing factors. Finally, cell-fusion experiments have shown that some hnRNP proteins remain localized in the nucleus, whereas others cycle in and out of the cytoplasm, suggesting that they function in the transport of mRNA. We discuss the role of these proteins in nuclear transport in Section 12.3.

3' Cleavage and Polyadenylation of Pre-mRNAs Are Tightly Coupled

In eukaryotic cells, all mRNAs, except histone mRNAs, have a 3' poly(A) tail. Early studies of pulse-labeled adenovirus and SV40 RNA demonstrated that the viral primary transcripts extend beyond the site in the viral mRNAs from which the poly(A) tail extends. These results suggested that A residues are added to a 3' hydroxyl generated by endonu-cleolytic cleavage of a longer transcript, but the predicted downstream RNA fragments never were detected in vivo, presumably because of their rapid degradation. That cleavage of a primary transcript precedes its polyadenylation was firmly established by detection of both predicted cleavage

► FIGURE 12-4 Model for cleavage and polyadenylation of pre-mRNAs in mammalian cells. Cleavage and polyadenylation specificity factor (CPSF) binds to the upstream AAUAAA poly(A) signal. CStF interacts with a downstream GU- or U-rich sequence and with bound CPSF, forming a loop in the RNA; binding of CFI and CFII help stabilize the complex. Binding of poly(A) polymerase (PAP) then stimulates cleavage at a poly(A) site, which usually is 10-35 nucleotides 3' of the upstream poly(A) signal. The cleavage factors are released, as is the downstream RNA cleavage product, which is rapidly degraded. Bound PAP then adds =12 A residues at a slow rate to the 3'-hydroxyl group generated by the cleavage reaction. Binding of poly(A)-binding protein II (PABPII) to the initial short poly(A) tail accelerates the rate of addition by PAP After 200-250 A residues have been added, PABPII signals PAP to stop polymerization.

signal Poly(A) site signal


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