Molecular architecture of the human pre-mRNA 3' processing complex

Abstract

Pre-mRNA 3' end formation is an essential step in eukaryotic gene expression. Over half of human genes produce alternatively polyadenylated mRNAs, suggesting that regulated polyadenylation is an important mechanism for posttranscriptional gene control. Although a number of mammalian mRNA 3' processing factors have been identified, the full protein composition of the 3' processing machinery has not been determined, and its structure is unknown. Here we report the purification and subsequent proteomic and structural characterization of human mRNA 3' processing complexes. Remarkably, the purified 3' processing complex contains approximately 85 proteins, including known and new core 3' processing factors and over 50 proteins that may mediate crosstalk with other processes. Electron microscopic analyses show that the core 3' processing complex has a distinct "kidney" shape and is approximately 250 A in length. Together, our data has revealed the complexity and molecular architecture of the pre-mRNA 3' processing complex.

Figure 1. Characterization of RNA substrates

(A) A schematic drawing of the RNA substrates. 3MS2 tags (hairpins) and the AAUAAA element in wild-type substrates and AACAAA in mutant substrates (boxes) are shown. The site of the single nucleotide change is marked with an asterisk. (B) Comparison of the wild type and mutant RNA substrates in polyadenylation and complex formation assays. A time course of polyadenylation reactions was analyzed on a denaturing 6% gel (top), or on a 1.5% native agarose gel (bottom), and visualized by phosphorimaging. (C) RNA profiles (measured by radioactivity) on glycerol gradients. The peaks corresponding to the 30S/H and 50S/P complexes, and the peak positions of E. coli 30S and 50S ribosomes from a parallel gradient, are marked.

Figure 2. Purification of 3′ processing complexes

(A) RNAs from input, and purified 30S/H and 50S/P complexes were purified, resolved and visualized as described above. (B and C) Proteins from the purified 30S/H and 50S/P complexes were resolved by SDS-PAGE and analyzed by silver staining (MBP-MS2 and several western-confirmed bands are labeled) (B) and western blotting (C).

Figure 3. Purified 3′ processing complexes are functional in a complementation assay

Purified 3M-SVL P complexes alone (P, lane 1), supplemented with buffer D (Bfr, lane 2), or increasing amounts of CF fraction (CF, lanes 3-5) were incubated under cleavage conditions. Standard cleavage assays with NE and CF are shown as controls.

Figure 4. WDR33 is a bona fide component of the CPSF complex

(A and B) Chacterization of the immuopurified CPSF73 complex. Proteins from Flag-IP of NE from HEK293 or the CPSF73-3Flag cell line were resolved by SDS-PAGE and stained with silver (A), or analyzed by Western blotting using specific antibodies (B). Protein bands in (A) whose identities were confirmed by western are labeled. (C) Immunopurified CPSF complexes were analyzed by gel filtration using a Superdex-200 column. Proteins from input and each fraction were analyzed by SDS-PAGE and silver staining (top panel) and western (bottom panel). WDR33 is marked by a arrow. (D) Western analyses of the mock-treated (mock) or WDR33-depleted (ΔWDR33) NE. Intensities of individual bands were quantified using the Odyssey Infrared scanner (Li-Cor). (E) WDR33 is essential for 3′ processing. Mock, ΔWDR33 NE alone, or ΔWDR33 supplemented with immunopurified CPSF were used in standard cleavage assays. RNAs were resolved and visualized as described above.

Figure 5. PP1 is required for polyadenylation, but not for cleavage

(A) Western analyses of mock-treated (Mock) or microcystin-treated (MC) NE. (B) Depletion of PP1/2A phosphatases has no effect on cleavage. Mock and MC NE were used in standard in vitro cleavage assays with SVL as the substrate. Pre-mRNA and the 5’ cleaved products are marked. (C) PP1 is required for polyadenylation. Mock, MC NE alone, or MC NE supplemented with recombinant PP1 were used in standard polyadenylation assays with SVL as the substrate. Pre-mRNA and poly(A)+ RNAs are marked.

Figure 6. EM analyses of the purified 3′ processing complex

(A) A typical CCD micrograph of the negatively stained purified 3′ processing complexes. Bar, 100 nm. (B) A gallery of representative particles. Bar, 20 nm.