THE C-PROTEIN TETRAMER BINDS 230 TO 240 NUCLEOTIDES OF PREMESSENGER RNA AND NUCLEATES THE ASSEMBLY OF 40S HETEROGENEOUS NUCLEAR RIBONUCLEOPROTEIN-PARTICLES

A series of in vitro protein-RNA binding studies using purified native (C1)3C2 and (A2)3B1 tetramers, total soluble heterogeneous nuclear ri bonucleoprotein (hnRNP), and pre-mRNA molecules differing in length an d sequence have revealed that a single C-protein tetramer has an RNA s ite size of 230 to 240 nucleotides (nt). Two tetramers bind twice this RNA length, and three tetramers fold monoparticle lengths of RNA (700 nt) into a unique 19S triangular complex. In the absence of this uniq ue structure, the basic A- and B-group proteins bind RNA to form sever al different artifactual structures which are not present in preparati ons of native hnRNP and which do not function in hnRNP assembly. Three (A2)3B1 tetramers bind the 19S complex to form a 35S assembly interme diate. Following UV irradiation to immobilize the C proteins on the pa ckaged RNA, the 19S triangular complex is recovered as a remnant struc ture from both native and reconstituted hnRNP particles. C protein-RNA complexes composed of three, six, or nine tetramers (one, two, or thr ee triangular complexes) nucleate the stoichiometric assembly of monom er, dimer, and trimer hnRNP particles. The binding of C-protein tetram ers to RNAs longer than 230 nt is through a self-cooperative combinato rial mode. RNA packaged in the 19S complex and in 40S hnRNP particles is efficiently spliced in vitro. These findings demonstrate that forma tion of the triangular C protein-RNA complex is an obligate first even t in the in vitro and probably the in vivo assembly the 40S hnRNP core particle, and they provide insight into the mechanism through which t he core proteins package 700-nt increments of RNA. These findings also demonstrate that unless excluded by other factors, the C proteins are likely to be located along the length of nascent transcripts.