What we’re talking about

A recent paper from James Manley’s lab details a proteomic analysis of the polyadenylation complex.

In this study, the polyadenylation complex was purified using an affinity technique, attaching a functional polyadenylation signal to a series of MS2 coat protein binding sites (of course, all within the context of an RNA), incubating this RNA with Hela cell extracts, and purifying the RNA using a maltose binding protein-MS2 coat protein fusion. The protein components of the complex were characterized using the so-called Multidimensional Protein Identification Technology (MudPIT). In addition to the usual players* (CPSF, CstF, CFIm) were found proteins suggestive of links with transcription, splicing, and DNA repair. Curiously, the sole poly(A) polymerase found was not the canonical enzyme but an isoform first identified as an enzyme that adenylates the RNA present in the so-called Signal Recognition Particle (SRP). Moreover, readily detectable was a testis-specific CstF64 variant (CstF64-tau) as well as the canonical 64 kD subunit of CstF. CFIIm was largely absent.

A summing up of the protein subunits indicates that the core complex is not much smaller than the bacterial large ribosomal subunit; the pictures shown below are electron micrographs that show as much, and reveal a distinctive shape for the complex. Lots of pieces remain to be filled in – how does the dimeric nature of CstF77 fit into this structure, how much other redundancy is there, how flexible or dynamic is the complex, etc.? Regardless, it’s fun to see the object of one’s intense interest.

The money quote, not a quote but rather a snapshot:

(from the paper by Shi et al. (2008)) 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

The 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 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 250 Å in length. Together, our data has revealed the complexity and molecular architecture of the pre-mRNA 3 processing complex.”

Shi Y, Di Giammartino DC, Taylor D, Sarkeshik A, Rice WJ, Yates JR 3rd, Frank J, Manley JL. Molecular architecture of the human pre-mRNA 3′ processing complex. Mol Cell. 2009 Feb 13;33(3):365-76.

* this figure illustrates the players (CPSF, CstF, etc.) mentioned here. The associated review describes them in more detail.