Distinct RNP complexes of shuttling hnRNP proteins with pre-mRNA and mRNA: candidate intermediates in formation and export of mRNA

Nascent pre-mRNAs associate with hnRNP proteins in hnRNP complexes, the natural substrates for mRNA processing. Several lines of evidence indicate that hnRNP complexes undergo substantial remodeling during mRNA formation and export. Here we report the isolation of three distinct types of pre-mRNP and mRNP complexes from HeLa cells associated with hnRNP A1, a shuttling hnRNP protein. Based on their RNA and protein compositions, these complexes are likely to represent distinct stages in the nucleocytoplasmic shuttling pathway of hnRNP A1 with its bound RNAs. In the cytoplasm, A1 is associated with its nuclear import receptor (transportin), the cytoplasmic poly(A)-binding protein, and mRNA. In the nucleus, A1 is found in two distinct types of complexes that are differently associated with nuclear structures. One class contains pre-mRNA and mRNA and is identical to previously described hnRNP complexes. The other class behaves as freely diffusible nuclear mRNPs (nmRNPs) at late nuclear stages of maturation and possibly associated with nuclear mRNA export. These nmRNPs differ from hnRNPs in that while they contain shuttling hnRNP proteins, the mRNA export factor REF, and mRNA, they do not contain nonshuttling hnRNP proteins or pre-mRNA. Importantly, nmRNPs also contain proteins not found in hnRNP complexes. These include the alternatively spliced isoforms D01 and D02 of the hnRNP D proteins, the E0 isoform of the hnRNP E proteins, and LRP130, a previously reported protein with unknown function that appears to have a novel type of RNA-binding domain. The characteristics of these complexes indicate that they result from RNP remodeling associated with mRNA maturation and delineate specific changes in RNP protein composition during formation and transport of mRNA in vivo.     

Characterization and primary structure of the poly(C)-binding heterogeneous nuclear ribonucleoprotein complex K protein.

At least 20 major proteins make up the ribonucleoprotein (RNP) complexes of heterogeneous nuclear RNA (hnRNA) in mammalian cells. Many of these proteins have distinct RNA-binding specificities. The abundant, acidic heterogeneous nuclear RNP (hnRNP) K and J proteins (66 and 64 kDa, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) are unique among the hnRNP proteins in their binding preference: they bind tenaciously to poly(C), and they are the major oligo(C)- and poly(C)-binding proteins in human HeLa cells. We purified K and J from HeLa cells by affinity chromatography and produced monoclonal antibodies to them. K and J are immunologically related and conserved among various vertebrates. Immunofluorescence microscopy with antibodies shows that K and J are located in the nucleoplasm. cDNA clones for K were isolated, and their sequences were determined. The predicted amino acid sequence of K does not contain an RNP consensus sequence found in many characterized hnRNP proteins and shows no extensive homology to sequences of any known proteins. The K protein contains two internal repeats not found in other known proteins, as well as GlyArgGlyGly and GlyArgGlyGlyPhe sequences, which occur frequently in many RNA-binding proteins. Overall, K represents a novel type of hnRNA-binding protein. It is likely that K and J play a role in the nuclear metabolism of hnRNAs, particularly for pre-mRNAs that contain cytidine-rich sequences.     

Determination of the RNA binding specificity of the heterogeneous nuclear ribonucleoprotein (hnRNP) H/H'/F/2H9 family

Members of the heterogeneous nuclear ribonucleoprotein (hnRNP) H protein family, H, H', F, and 2H9, are involved in pre-mRNA processing. We analyzed the assembly of these proteins from splicing extracts onto four RNA regulatory elements as follows: a high affinity hnRNP A1-binding site (WA1), a sequence involved in Rev-dependent export (p17gag INS), an exonic splicing silencer from the beta-tropomyosin gene, and an intronic splicing regulator (downstream control sequence (DCS) from the c-src gene. The entire family binds the WA1, instability (INS), and beta-tropomyosin substrates, and the core-binding site for each is a run of three G residues followed by an A. Transfer of small regions containing this sequence to a substrate lacking hnRNP H binding activity is sufficient to promote binding of all family members. The c-src DCS has been shown to assemble hnRNP H, not hnRNP F, from HeLa cell extracts, and we show that hnRNP 2H9 does not bind this element. The DCS contains five G residues followed by a C. Mutation of the C to an A changes the specificity of the DCS from a substrate that binds only hnRNP H/H' to a binding site for all hnRNP H family members. We conclude that the sequence GGGA is recognized by all hnRNP H family proteins.     

多种功能的poly(C)-结合蛋白

细胞内的RNA一般不会单独存在,而是与各种各样的RNA结合蛋白（RBPs）绑定在一起,形成核糖核蛋白复合体（RNP complexes）影响着RNA的加工与转归. Poly(C) 结合蛋白是一类重要的RNA结合蛋白,可分为两组：hnRNP K 和PCBP1 4. 它们以序列特异的方式与核酸嘧啶富含区相结合. 这类蛋白具有共同的结构模体（motif）,即hnRNP K 同源（KH）域. KH域是与mRNA结合的结构基础,也是机体内调控系统的组成部分,可使得Poly(C) 结合蛋白参与蛋白/核酸、蛋白/蛋白之间的相互作用,范围涉及复制、转录、mRNA稳定和翻译控制过程等. 对Poly(C) 结合蛋白功能的深刻认识可使我们洞察多种疾病的病理生理过程.     

Cytosine-block telomeric type DNA-binding activity of hnRNP proteins from human cell lines

Following the observation of the presence in mammalian nuclear extracts of a DNA binding activity quite specific for the single-stranded C-rich telomeric motif, we have isolated from the K562 human cell line by affinity chromatography and identified by mass spectrometry a number of proteins able to bind to this sequence. All of them belong to different heterogeneous nuclear ribonucleoprotein subgroups (hnRNP). Whereas many of them, namely hnRNP K, two isoforms of hnRNP I, and the factor JKTBP, appear to bind to this sequence with limited specificity after isolation, an isoform of hnRNP D (alias AUF1) and particularly hnRNP E1 (alias PCBP-1) show a remarkable specificity for the (CCCTAA)n repeated motif. Both have been obtained also as recombinant proteins expressed in Escherichia coli and have been shown to retain their binding specificity toward the C-block repeated sequence. In the light of the current knowledge about these proteins, their possible involvement in telomere functioning is discussed.     

The hnRNP F protein: unique primary structure, nucleic acid-binding properties, and subcellular localization.

More than 20 different heterogeneous nuclear ribonucleoproteins (hnRNPs) are associated with pre-mRNAs in the nucleus of mammalian cells and these proteins appear to influence pre-mRNA processing and other aspects of mRNA metabolism and transport. The arrangement of hnRNP proteins on pre-mRNAs is likely to be unique for each RNA and may be determined by the different RNA-binding preferences of each of these proteins. hnRNP F (M(r) = 53 kD, pI = 6.1) and hnRNP H (M(r) = 56 kD, pI = 6.7-7.1) are abundant components of immunopurified hnRNP complexes and they have distinct nucleic acid binding properties. Unlike other hnRNP proteins which display a varying range of affinities for different ribonucleotidehomopolymers and ssDNA, hnRNP F and hnRNP H bind only to poly(rG) in vitro. hnRNP F and hnRNP H were purified from HeLa cells by poly(rG) affinity chromatography and oligonucleotides derived from peptide sequences were used to isolate a cDNA encoding hnRNP F. The predicted amino acid sequence of hnRNP F revealed a novel protein with three repeated domains related to the RNP consensus sequence RNA-binding domain. Monoclonal antibodies produced against bacterially expressed hnRNP F were specific for both hnRNP F and hnRNP H and recognized related proteins in divergent organisms, including in the yeast Saccharomyces cerevisiae. hnRNP F and hnRNP H are thus highly related immunologically and they share identical peptides. Interestingly, immunofluorescence microscopy revealed that hnRNP F and hnRNP H are concentrated in discrete regions of the nucleoplasm, in contrast to the general nucleoplasmic distribution of previously characterized hnRNP proteins. The unique RNA-binding properties, amino acid sequence and distinct intranuclear localization of hnRNP F and hnRNP H make them novel hnRNP proteins that are likely to be important for the processing of RNAs containing guanosine-rich sequences.