CFIm – even more reducibility

Earlier, I discussed interesting results indicating that the canonical Cleavage and Polyadenylation Specificity Factor (or CPSF) functions in the 3′ processing of histone mRNAs, a reaction that involves a subset of the proteins associated with mRNA 3′ end formation and polyadenylation. One developing idea I mentioned was that the polyadenylation complex may consist of a core that functions in other processes (such as histone mRNA 3′ end formation), and that its activity is modulated by a varying suite of accessory factors.  Recently, a study appeared that elaborates on this finding.  Briefly, Ruepp et al. have found that the 68 kD subunit of the so-called mammalian Cleavage Factor I (CFIm68, for short) associates with the U7 snRNP complex, and functions in histone mRNA 3′ processing.  As interestingly, they report that this subunit does not act with its partner in the nuclear polyadenylation complex, the 25 kD subunit of CFIm (CFIm25).

CFIm hasn’t been much discussed on this blog, largely because, until recently, its status in plants has been very murky (to me, at least).  However, thanks to a pleasant and very fruitful chat with Greg Gilmartin at the RNA 2010 Meeting, this cloud in my mind has been largely dissipated.  Thus, plants seem to possess orthologs for both subunits of CFIm (the 68 kD subunit being one of the “absent” proteins in our 2008 paper).

CFIm is a fascinating factor – it is a heterodimer* that binds RNA sequences upstream from the canonical AAUAAA motif and can enhance usage of AAUAAA variants that by themselves are inefficient.  The relationship between CFIm and AAUAAA brings to mind that between the far-upstream element and near-upstream element in the plant polyadenylation signal; the sequence preferences for CFIm are not unlike some of the motifs associated with FUEs, and the NUE has been suggested to be the equivalent of AAUAAA.  (I don’t know how accurate the analogy is, but the parallels are thought-provoking.)  The interplay between CFIm and weak AAUAAA-like motifs may be important in the sorts of alternative polyadenylation that are associated with cellular growth, differentiation, and cancer.  Given these connections, the plant CFIm counterpart (whatever it really looks like) stands to be an important player in the some pretty interesting phenomena.

So, what does all this have to do with Ruepp et al?  Who knows, to be honest.  But one theme that Ruepp et al. revisits is the idea that the 3′ processing complexes are actually a multitude of variants, each consisting of a common core along with a different suite of accessory subunits.  The finding that CFIm68 can function apart from CFIm25 adds a subtle twist to this theme.  Whether or not a similar situation exists in plants is an open question.  But what is becoming ever more clear is that the polyadenylation complex is actually quite a plastic entity. This plasticity is very possibly a key to the regulation that is linked with the plant polyadenylation complex.

The abstract from the paper:

Metazoan replication-dependent histone pre-mRNAs undergo a unique 3′-cleavage reaction which does not result in mRNA polyadenylation. Although the cleavage site is defined by histone-specific factors (hairpin binding protein, a 100-kDa zinc-finger protein and the U7 snRNP), a large complex consisting of cleavage/polyadenylation specificity factor, two subunits of cleavage stimulation factor and symplekin acts as the effector of RNA cleavage. Here, we report that yet another protein involved in cleavage/polyadenylation, mammalian cleavage factor I 68-kDa subunit (CF I(m)68), participates in histone RNA 3′-end processing. CF I(m)68 was found in a highly purified U7 snRNP preparation. Its interaction with the U7 snRNP depends on the N-terminus of the U7 snRNP protein Lsm11, known to be important for histone RNA processing. In vivo, both depletion and overexpression of CF I(m)68 cause significant decreases in processing efficiency. In vitro 3′-end processing is slightly stimulated by the addition of low amounts of CF I(m)68, but inhibited by high amounts or by anti-CF I(m)68 antibody. Finally, immunoprecipitation of CF I(m)68 results in a strong enrichment of histone pre-mRNAs. In contrast, the small CF I(m) subunit, CF I(m)25, does not appear to be involved in histone RNA processing.

The citation:

Ruepp MD, Vivarelli S, Pillai RS, Kleinschmidt N, Azzouz TN, Barabino SM, Schümperli D. The 68 kDa subunit of mammalian cleavage factor I interacts with the U7 small nuclear ribonucleoprotein and participates in 3′-end processing of animal histone mRNAs. Nucleic Acids Res. 2010 Jul 15. [Epub ahead of print] PubMed PMID: 20634199.

* – the CFIm heterodimer is actually a bit more complicated than this, as it is actually a collection of heterodimers that consist of CFIm25 and one of three (similar) larger proteins, of MW 59, 68, or 72 kD.

6 Responses to CFIm – even more reducibility

  1. Clem Weidenbenner says:

    Art said:
    But one theme that Ruepp et al. revisits is the idea that the 3′ processing complexes are actually a multitude of variants, each consisting of a common core along with a different suite of accessory subunits.

    Do you have a feel for whether these various complexes are built within a cell and once built remain fairly static OR could individual cores have ancillary subunits added or subtracted in order to take on different responsibilities?

  2. Arthur Hunt says:

    Hi Clem,

    That’s a great question. I think that I would lean to the second possibility – after all, the complex resolves easily during standard biochemical fractionations, and the stoichiometries of some subunits often is not exactly 1:1 (or whatever). And I have probably suggested as much in some recent reviews (and perhaps papers). But the first possibility has to be considered seriously. As should combinations of these possibilities, that may also involve the transciption and splicing machineries (and more).

    Now I’m going to be up nights figuring out (in my head) ways to ask these questions experimentally ….

  3. Clem Weidenbenner says:

    No problems, flourescent labeling to the rescue!!. You already know most of the players…and even which particular players are likely recruited for specific jobs. Tag those bad boys and start filming. Post the video here and start writing your Nobel acceptance speech.

    Seroiusly, as one can label a few different targets at a time (with different colors) this might be worth rolling around in the mind.

  4. Clem Weidenbenner says:

    A little off the present thread, but have you ever written here about lncRNAs or uORFs?

    There is a perspectives piece in the 16 July issue of Science that discusses these. Rosenberg and Desplan, (2010)Sci. 329: 284-285. Everything I’ve seen on these so far is from the animal kingdom, but I’ve not gone very far into this yet.

  5. Arthur Hunt says:

    Hi Clem,

    The fluorescent tagging suggestion is intriguing. I am not sure it has the resolution for the contrasting models I mentioned, but there may be possibilities.

    As for lncRNAs and uORFs, plant scientists have been familiar with such things for a long time. For example, I can recall when enod40 was supposed to be a noncoding RNA, then when it was discovered to be a uORF gene, then when it was shown to act as both a small protein-coding gene and a functional RNA. This story started to unfold in the mid-1990’s.

  6. Clem Weidenbenner says:

    Thanks for the enod40 lead… looked it up and ran across something else I’m interested in as well.

    As for resolution of fluorescent tags – the technology is moving along by leaps and bounds. A limitation may come from there being *too many* targets. How many of these complexes will there be in an Arabidopsis cell?

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