Ribonucleoproteins package 700 nucleotides of pre-mRNA into a repeating array of regular particles.
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 A1 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.
Conway
G
G
Department of Molecular Biology, Vanderbilt University, Nashville, Tennessee 37235.
Wooley
J
J
Bibring
T
T
LeStourgeon
W M
WM
D006367:HeLa Cells; D006801:Humans; D008854:Microscopy, Electron; D010316:Particle Size; D012322:RNA Precursors / Q000032:analysis / Q000378:metabolism*; D012326:RNA Splicing; D012261:Ribonucleoproteins / Q000032:analysis / Q000378:metabolism*
D012322:RNA Precursors; D012261:Ribonucleoproteins