Ribonucleoproteins package 700 nucleotides of pre-mRNA into a repeating array of regular particles.

Abstract

An assay for the in vitro assembly of HeLa cell 40S nuclear ribonucleoprotein particles (hnRNP particles) has been developed. The substrates were single-stranded nucleic acid polymers of defined length and sequence prepared in vitro and the six major core particle proteins from isolated 40S hnRNP. The fidelity of in vitro assembly was evaluated on various physical parameters, including sedimentation, salt dissociation, polypeptide stoichiometry, UV-activated protein-RNA cross-linking, and overall morphology. Correct particle assembly depended on RNA length and on the input protein/RNA ratio but not on the concentration of the reactant mixture nor on the presence or absence of internal RNA processing signals, a 5'-cap structure, a 3'-poly(A) moiety, or ATP as energy source. RNA lengths between 685 and 726 nucleotides supported correct particle assembly. Dimers and oligomeric complexes that possessed the same polypeptide stoichiometry as native hnRNP assembled on RNA chains that were integral multiples of 700 nucleotides. Intermediate-length RNA supported the assembly of nonstoichiometric complexes lacking structural homogeneity. An analysis of these complexes indicates that proteins Al and A2 may be the first proteins to bind RNA during particle assembly. We conclude that the major proteins of 40S hnRNP particles contain the necessary information for packaging nascent transcripts into a repeating "ribonucleosomal" structure possessing a defined RNA length and protein composition but do not themselves contain the information for modulating packaging that may be required for RNA splicing.The biochemical events of pre-mRNA processing are known largely through studies on the RNA intermediates produced during splicing and studies on the nucleotide sequences required for RNA maturation (for reviews, see references 17 and 42). Nascent transcripts, however, are packaged by a specific subset of major nuclear proteins during transcription to form a ribonucleoprotein (RNP) complex in which the events of RNA splicing occur (see references 8 and 10 for reviews). Historically, these complexes were termed heterogeneous nuclear ribonucleoprotein particles (hnRNP) because the packaged moiety was heterogeneous nuclear RNA and not because the individual particles were known to be heterogeneous in composition and structure (10,38,41). Several observations indicate that nascent transcripts are in fact packaged into a regular repeating structure composed of a contiguous array of 40S hnRNP complexes. These observations suggest that the complexes may play only a passive role in RNA splicing. Other observations suggest that packaging may be transcript specific and thus imply that hnRNP may play a more direct role in the events of RNA processing.Observations in support of a fundamental packaging function for hnRNP are the following. (i) The intranuclear concentration of the major hnRNP is high. For example, in actively growing mammalian cells there is 20 to 30% as much individual core particle hnRNP as individual core particle histone (...

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

“…Surprisingly, an immunologically related protein of approximately the same molecular mass as hnRNP F was detected in the yeast S. cerevisiae. hnRNP proteins are commonly conserved among vertebrates, however few related proteins have been identified in yeast (1,29 Although it has been demonstrated that many hnRNP proteins display RNA sequence binding preferences, all of the previously characterized proteins appear to also bind nucleic acids in an apparently sequence independent manner (2,41,42). One unique feature of hnRNP F and hnRNP H is that poly(rG) is the only nucleic acid that they have been found to bind to in vitro (2,28).…”

General splicing factors SF2 and SC35 have equivalent activities in vitro, and both affect alternative 5' and 3' splice site selection.

“…The equilibrium binding of hnRNP Al to single-stranded polyaucleotides is cooperative and shows limited base specificity (28). In vitro reconstitution studies with hnRNP core proteins also show a lack of sequence specificity (29). However, the contacts ofhnRNP Al with pre-mRNA are sensitive to the presence of additional factors in nuclear extracts (30), and specific binding of hRNP Al …”

Characterization and molecular cloning of polypyrimidine tract-binding protein: a component of a complex necessary for pre-mRNA splicing.

“…However, this protein binds to poly(U)-Sepharose in the presence of 2 M KC1, whereas PTB elutes from this resin at 750 mM KC1, suggesting that these proteins are distinct. Moreover, hnRNP proteins are generally thought to bind RNA nonspecifically (Conway et al 1988), much the way histones bind DNA. If this is the case, PTB does not fit the description of an hnRNP protein.…”