| The 3' end formation of most eukaryotic messenger RNA precursors (pre-mRNAs) involves two reactions, an endonuclease cleavage followed by polyadenylation. A large number of protein factors have been identified as members of 3' processing machinery. Among them is cleavage stimulation factor (CstF), a heterotrimeric protein complex essential for both the efficiency and the specificity of the cleavage reaction. Its largest subunit, CstF-77, can mediates both the assembly of the CstF complex and the interactions with other protein factors in the 3' processing machinery. Here we determined the crystal structure of the HAT (h&barbelow;alf a&barbelow; T&barbelow;PR) domain of murine CstF-77, as well as its C-terminal sub-domain, the HAT-C domain. Our studies showed that the HAT domain of CstF-77 is formed of two distinct sub-domains, the HAT-N and the HAT-C sub-domains. Moreover, CstF-77 forms a homodimer mediated by the HAT-C sub-domain. This dimerization event has been further verified by various methods including static light-scattering studies, yeast two-hybrid assays, analytical ultracentrifugation measurements, and small angle X-ray scattering experiments. Our studies supported a dimeric association of the CstF complex in pre-mRNA 3' processing machinery.;Although a lot has been learned about pre-mRNA 3' end formation in the nucleus, a similar process in the mitochondria is much less understood. To this end, we conducted structural and functional studies on human mitochondria poly(A) polymerase, hPAPD1. This protein shares high structural similarity to that of the canonical PAPs for having similar palm domain (the catalytic domain) and fingers domain. However, hPAPD1 lacks the RNA binding domain which resides on the C-terminus of canonical PAPs, making it more similar to a cytoplasmic PAP, GLD-2. In the crystal structure of hPAPD1, we observed a unique N-terminal domain, which involves in the dimerization of this protein. Neither this domain nor the dimeric association has been reported for other PAPs before. In our following polyadenylation assays, we found that dimerization is critical for the activity of hPAPD1. Electrophoretic mobility shift assays (EMSA) also indicated this dimeric association may be involved in the recruitment of substrate RNA. |
