The C-protein tetramer binds 230 to 240 nucleotides of pre-mRNA 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 ribonucleoprotein (hnRNP), and pre-mRNA molecules differing in length and sequence have revealed that a single C-protein tetramer has an RNA site 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 unique structure, the basic A- and B-group proteins bind RNA to form several different artifactual structures which are not present in preparations of native hnRNP and which do not function in hnRNP assembly. Three (A2)3B1 tetramers bind the 19S complex to form a 35S assembly intermediate. Following UV irradiation to immobilize the C proteins on the packaged RNA, the 19S triangular complex is recovered as a remnant structure from both native and reconstituted hnRNP particles. C protein-RNA complexes composed of three, six, or nine tetramers (one, two, or three triangular complexes) nucleate the stoichiometric assembly of monomer, dimer, and trimer hnRNP particles. The binding of C-protein tetramers to RNAs longer than 230 nt is through a self-cooperative combinatorial mode. RNA packaged in the 19S complex and in 40S hnRNP particles is efficiently spliced in vitro. These findings demonstrate that formation of the triangular C protein-RNA complex is an obligate first event in the in vitro and probably the in vivo assembly the 40S hnRNP core particle, and they provide insight into the mechanism through which the 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.

Characterization of genes which are transiently expressed during the preaggregative phase of development of Dictyostelium discoideum.

We have identified and characterized three genes, the I genes (I for induced), which are induced during the preaggregative phase of the developmental program of Dictyostelium discoideum. None of these genes are expressed in cells growing vegetatively on bacteria or in axenic broth, and their induction during early development is due to transcriptional activation. Developmental expression of I6, I8, and I11 occurs even in the absence of protein synthesis. Their induction is very rapid and occurs essentially at the onset of development. The expression is transient, peaking between 2 and 4 hr followed by a rapid loss of expression. These characteristics suggest that the induction of I6, I8, and I11 is a primary result of the initiation of development, and thus they represent the first such genes isolated. Although their expression behavior shares these characteristics, examination of their expression under various conditions of development and in a variety of aggregation-deficient mutant strains reveals that the details of the regulation and developmental control of these three genes are distinct.