In this report, we asked whether the interaction between Yra1 and Pcf11 that links mRNA export with 3’ end formation, could also modulate assembly of the CF1A 3’ processing complex and cleavage-polyadenylation. Our results are consistent with a model where Yra1 and Clp1 compete for binding to a common partner, Pcf11 as predicted by Saguez and Jensen 37
(). Several lines of evidence support this model:1) Excess Clp1 can disrupt Yra1–Pcf11 complexes in vitro
(); 2) Pcf11 and Clp1 occupancy increases in the 3’ flanking region as Yra1 and Sub2 occupancy decreases () suggesting a concerted transfer of Yra1 and Sub2 from the polymerase complex onto the transcript 16,17
prior to poly(A) site cleavage; 3) Depletion of Yra1 in vivo
can enhance Clp1 occupancy relative to Pcf11 within the body of long genes (); 4) Full-length Yra1 inhibits cleavage and polyadenylation in vitro
whereas a fragment that does not bind Pcf11 has no inhibitory effect (). ChIP localization of Pcf11 and Clp1 suggests that these two factors can load onto the TEC separately. Whereas Pcf11 occupancy increases quite uniformly 5’–3’ across many ORFs 43,44
, Clp1 signals remain relatively low in the 5' region and then increase at the 3’ end (). We propose that Pcf11 and Yra1 may assemble into a “poised” complex on the TEC that is subsequently remodeled by Clp1 and Sub2 to achieve two important goals: 1) co-transcriptional assembly of CF1A through formation of the Clp1–Pcf11 contact and 2) transfer of Yra1 to the transcript ().
Unexpectedly Yra1 and Sub2 are also enriched on genes for CUTs, SUTs, snoRNAs and tRNAs (, Supplementary Fig. 2b, c
) where Pcf11 is also present 43
. Yra1 and Sub2 may promote export of ncRNAs, as they do for mRNAs. In any event, our results suggest that the function of these two factors is not confined to mRNA metabolism.
A dynamic balance between Pcf11–Yra1 and Pcf11–Clp1 complexes could influence the decision between alternative poly(A) sites. This idea is supported by the fact that depletion of Yra1 alters poly(A) site selection at numerous coding and non-coding RNAs (, , Supplementary Fig. 5b, c
). The heterogeneity of poly(A) site use at numerous yeast genes 41
is therefore not a purely stochastic phenomenon, but can be influenced by the export factor Yra1. Another yeast export factor, Npl3, has been shown to alter cleavage-polyadenylation by masking a cryptic processing site in the GAL7
3’ UTR 49
. Similarly many metazoan RNA binding proteins affect 3' end processing by competing with core cleavage-polyadenylation factors23,50,51
. Depletion of Yra1 does not simply activate cryptic sites, rather it can either enhance or suppress utilization of poly(A) sites in different contexts (–, Supplementary Fig. 5b, c
). We propose Yra1 influences poly(A) site choice by a regulated assembly mechanism in which interaction with Pcf11 regulates co-transcriptional formation of fully functional CF1A complexes (). This model also suggests that, like Yra1, Sub2 could affect poly(A) site choice. Our results do not rule out the possibility that Yra1 might influence 3’ end processing after it has been transferred to the nascent RNA and it remains possible that Yra1 modulates poly(A) site choice by multiple mechanisms.
We do not fully understand why Yra1 depletion affects poly(A) sites in different ways, but one distinction between Yra1-sensitive and -insensitive sites lies in the positioning of the EE motif that binds CF1B. Yra1-insensitive poly(A) sites have well-positioned EE motifs whereas at Yra1-sensitive sites, they are less precisely positioned relative to the cleavage site (). We speculate that at such non-optimal poly(A) sites, interaction of CF1A and CF1B with one another and with the RNA 28
may be more sensitive to modulators such as Yra1. How does Yra1 affect the selection of poly(A) sites at these “sensitive” sites? It could delay formation of fully active CF1A complexes by competing with Clp1, thereby suppressing use of particular poly(A) sites. This mechanism is consistent with the effects of Yra1 depletion on genes like ACT1
where poly(A) sites closer to the 5' end are favored (, , ). It is also possible that Yra1 could enhance formation of properly folded CF1A complexes by acting as a chaperone. This possibility is suggested by the fact that Aly, the human homologue of Yra1, has chaperone activity that enhances the DNA binding of several transcription factors 52,53
. Such a scenario could explain why processing at some sites is suppressed by Yra1 depletion. At first glance a positive influence on co-transcriptional CF1A assembly is not consistent with inhibition of cleavage-polyadenylation by Yra1 in vitro
(), however this experiment does not assay transcription-coupled processing, which may be affected differently than uncoupled processing. If different poly(A) sites in a transcription unit compete for processing by CF1A, then an increase or a decrease in the rate of assembly of the complex when Yra1 is depleted could shift the balance such that some sites are used more frequently and competing sites are used less frequently.
Human Aly and Pcf11 interact with one another like their yeast homologues15
suggesting that Aly could be a modulator of poly(A) site choice in metazoans. Human Pcf11 is not found in the CstF complex with the homologues of Rna14 and Rna15, but instead is a subunit of cleavage factor IIm
); the other subunit is human Clp1 54
. We speculate that REF/Aly, like Yra1, may affect the huPcf11-huClp1 interaction, and in doing so perhaps alters the function of CFIIm
Modulation of alternative polyadenylation in metazoans is an important means of regulating gene expression 23,38
. One way that such regulation can be achieved is through altered expression of core components of the 3’ processing machinery. The archetypal example is control of immunoglobulin heavy chain poly(A) site choice by regulating expression of CstF64, the homologue of yeast Rna15 (ref. 55
). Another recently discovered mechanism works through U1snRNP inhibition of cleavage at cryptic poly(A) sites, probably by interacting with core cleavage-polyadenylation factors 56
. Our results suggest a related mechanism for APA regulation; namely that an export adaptor, which interacts with the core 3’ end processing machinery, but is not itself a cleavage-polyadenylation factor, can function as a general modulator of poly(A) site choice.