Serafín Piñol-Roma

Heterogeneous nuclear ribonucleoprotein particles and the pathway of mRNA formation

Abstract Heterogeneous nuclear ribonucleoprotein (hnRNP) particles, the structures that package hnRNA, are one of the major constituents of the nucleus. Recent work has led to the immunopurification of hnRNP particles and the identification of their proteins, and demonstrated a role for hnRNP proteins in mRNA splicing. The molecular cloning and sequencing of cDNAs for RNP proteins made possible the discovery of a conserved RNA-binding domain and a RNP consensus sequence.

hnRNP proteins:Localization and transport between the nucleus and the cytoplasm

The proteins of heterogeneous nuclear ribonucleoprotein (hnRNP) complexes are among the most abundant proteins in the nucleus. They bind nascent pre-mRNAs and remain associated with them through their nuclear processing into mRNA. Recent findings indicate roles for hnRNP proteins in the biogenesis of mRNA and reveal a surprising intracellular localization pathway for these proteins. Several of the hnRNP proteins shuttle continuously between the nucleus and the cytoplasm, and the reaccumulation of the exported hnRNP proteins in the nucleus occurs by a novel process that is dependent on transcription by RNA polymerase II. These findings suggest possible novel functions for hnRNP proteins in the cytoplasm and in the nucleocytoplasmic transport of mRNA.

Cell cycle-regulated phosphorylation of the pre-mRNA-binding (heterogeneous nuclear ribonucleoprotein) C proteins.

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 (C1, M(r) of 41,000; C2, M(r) 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 H1 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.

Ultraviolet-induced cross-linking of RNA to proteins in vivo

Publisher Summary This chapter describes methods for RNA–protein cross-linking in living cells as a means of identification of RNA-binding proteins in vivo . The chapter focuses on heterogeneous nuclear RNA (hnRNA)- and messenger RNA (mRNA)-binding proteins. The most stringent definition of genuine ribonucleoproteins (RNPs) is that these are proteins, which are bound directly to the RNA of interest in the living cell. UV cross-linking of RNP complexes takes advantage of the fact that UV light of sufficient intensity generates highly reactive species of RNA, which react virtually indiscriminately with molecules, including proteins, with which the RNA is in stable, direct contact. The cross-linked proteins can be released from the RNA by exhaustive RNase digestion and can be analyzed by gel-electrophoretic techniques. This method provides a powerful tool for the identification of bona fide RNP proteins. The fractionation of cells into nuclear and cytoplasmic components prior to the chromatographic step allows the distinction between hnRNA-containing RNP complexes (hnRNPs), from the nuclear fraction, and mRNAcontaining RNP complexes (mRNPs), from the cytoplasmic fraction.

Immunological methods for purification and characterization of heterogeneous nuclear ribonucleoprotein particles.

Publisher Summary Heterogeneous nuclear RNAs (hnRNAs) are associated in the cell with specific proteins to form hnRNA-ribonucleoprotein (hnRNP) complexes, also referred to as hnRNP particles. The immunopurification procedure has a number of advantages over methods traditionally used for studying hnRNP complexes such as sucrose gradient sedimentation of 30 S particles and photochemical cross-linking of proteins to RNA. The purity of the immunopurified hnRNP complexes is obvious by the absence of abundant nuclear proteins such as histones. Under the conditions described here, there are no significant amounts of other RNP proteins, such as those of small nuclear RNPs (snRNPs). The initial step in the hnRNP immunopurification procedure involves separation of the nuclear and cytoplasmic fractions, and the subsequent removal of nucleoli and insoluble chromatin from the nuclear fraction in order to generate what is operationally defined as the nucleoplasm. It is also essential to always include a control immunopurification with preimmune serum or immunoglobulins from the parent myeloma cell line used for the production of the hybridomas.

Purification and characterization of proteins of heterogeneous nuclear ribonucleoprotein complexes by affinity chromatography

Publisher Summary This chapter describes the role of affinity chromatography in the purification and characterization of proteins of heterogeneous nuclear ribonucleoprotein complexes. Affinity chromatographic methods for purification of proteins rely on the specificity and relatively high affinity of a protein for an immobilized substrate. Among the advantages of the large scale purification of hnRNP proteins by affinity chromatography on ssDNA are its relative rapidity and ease, its independence of the intactness of hnRNP particles during sample preparation, the lack of a need for radioactive labeling of the hnRNA for detection of the hnRNP-containing fractions, and the stability of the ssDNA column. Addition of a heparin wash (1.0 mg/ml in 100 mM NaC1) after the binding results in greater purity of the fractions, most probably owing to the competition for those proteins that bind to the column predominantly via ionic interactions with the DNA phosphate backbone. Adjustment of the nucleoplasm salt content to lower concentrations results in increased binding of a larger number of hnRNPs to the ribohomopolymers.