Termination of transcription is a critical mechanistic step in regulating gene expression, particularly for genes with multiple polyadenylation sites. Here we report that the SR/hnRNP protein Npl3 directly enhances the elongation rate of RNAP II. Although co-transcriptional recruitment of eukaryotic RNA binding proteins is now well established, to our knowledge a direct stimulation of elongation by SR or hnRNP proteins has not been previously reported. No such stimulation is seen with the RNA binding protein Rna15 or the truncated Npl3 RRM domain, arguing that the Npl3 effect is not due to non-specific interaction with RNA.
The data presented is consistent with a model where Npl3 functions in two ways: it directly interacts with phosphorylated serine 2 of the polymerase CTD to promote elongation and at the same time binds RNA to antagonize binding of the polyA/termination factor Rna15 () 
. This combination of activities resembles the l-related phage 82 anti-terminator Q82
that, together with the NusA protein, decreases pausing of the bacterial RNA polymerase and protects the RNA transcript from termination factors 
Two activities of Npl3 promote elongation.
Phosphorylation of Npl3 reduces its interaction with RNAP II and RNA. It is interesting to speculate that the targeted phosphorylation of Npl3 at 3′ ends of genes might help trigger termination. This model is supported by chromatin immunoprecipitation experiments where crosslinking of Npl3 was observed to decrease downstream of the polyA site 
, by the expression analysis presented here where mutations at S411 or in the second RRM of Npl3 were shown to specifically affect transcript signals at 3′ ends of genes, and by UV-crosslinking where the Cka1 phosphorylation decreased Npl3's competition for binding to polyA signals. In addition, a recent report by Lund et al
suggests that phosphorylation is involved the autoregulation of the NPL3
. The cytosolic kinase Sky1 phosphorylates Npl3 
. Thus far, no other kinase had been linked to the phosphorylation of Npl3. CK2 is the only kinase reported to phosphorylate proteins involved in mRNA 3′ end formation. Phosphorylation of 3′ end processing factors Pta1 and Fip1 by CK2 affects their ability to cleave and polyadenylate the mRNA 
. Therefore, the phosphorylation of Npl3 by CK2 shown here would support a targeted phosphorylation at the 3′ end. The existence of nuclear and cytosolic kinases that phosphorylate an mRNA shuttling protein at an identical residue suggests that the same phosphorylation switch, which functions by modulating protein-protein and -RNA interactions can be activated in different compartments of the cell. It remains to be determined what other kinases operate at the 3′ end or whether additional transcription-associated kinases can control the phosphorylation state of Npl3 to regulate its activity at sites of transcription.
The model postulates several events that would affect assembly of the polyA/termination machinery at the 3′ end ()
. First, upon dissociation of Npl3, the elongation rate of RNAP II would decrease to enhance the window of opportunity for polyA factors to bind to signals in the nascent RNA. It is known that the rate of transcription elongation can affect alternative splicing decisions 
, so it is reasonable to assume that a similar relationship can exist between elongation rate and polyadenylation. Interestingly, the elongation promoting activity of Npl3 is weaker in an RNA binding mutant and is inhibited by Rna15, which suggests that binding to the RNA transcript by Npl3 is important for stimulating RNAP II. Binding of Npl3 to the nascent transcript might stabilize interactions between the CTD, RNAP II and the RNA and prevent premature 3′ end formation. Accordingly, interactions between the CTD of the mammalian RNAP II with the RNA were shown to be critical to suppress premature termination 
. Additionally, binding of Npl3 to the RNA may prevent the formation of arrest-inducing secondary structures, or suppress pausing at cryptic polyA sites.
A second event upon dissociation of Npl3 would be a shift in the balance of the competition between Npl3 and Rna14/15 for RNA-binding. Several lines of evidence have shown that this competition is important for proper polyadenylation. Transcription readthrough of weakened polyA signals can be suppressed by mutation/deletion of Npl3 or Cbp20/80, or by overexpression of the polyadenylation factors Rna14 or Hrp1 
. This competition between Npl3 and Rna15 can be reconstituted in vitro 
, and here we show that this competition can be shifted by the CK2 phosphorylation of Npl3.
There are many examples of SR proteins being regulated by phosphorylation. In the case of Srp40, phosphorylation of the RS domain is required for sequence-specific RNA binding 
. For another RS protein, ASF/SF2, phosphorylation enhances contacts essential for splicing 
. The identification by mass spectrometry of multiple phosphorylated serines in vivo
and in vitro
suggests that Npl3 is hyperphosphorylated at the RRM and RGG/RS domains. In the case of Npl3-S411, its phosphorylation decreases crosslinking of the protein to RNA 
(this study), and also functions in autoregulation of the NPL3
. Our study suggests that CK2 is required for the phosphorylation of S411, and we also show that this residue appears to affect Npl3's interaction with RNAP II. Thus, S411 participates in the binding for both RNA and RNAP II.
While this manuscript was in preparation Lund et al
reported of an effect of Npl3 in autoregulation of its own transcript 
. In this study, phosphorylation of Npl3-S411 was observed to increase termination efficiency, thus modulating its own protein levels. Our expression analysis suggests that the termination of NPL3
represents a unique case likely shared by a limited number of genes. Previous work by Steinmetz and co-workers suggested that the choice of termination sites at the NPL3
locus involved a Sen1 anti-termination pathway 
. In addition, the phosphorylation decrease observed for S411 in the cka1
alleles reported in this study did not significantly affect the steady state levels of Npl3, as observed by Lund and co-workers. Therefore, we believe that the autoregulation of Npl3 is likely the result of an activity that is distinct from that mediated by CK2. At this time, the mechanism by which Npl3 functions in its autoregulation by allowing the selection of an alternative termination site remains unresolved.
Our study suggests that Npl3 affects elongation and termination in a high percentage of genes (~30%) making this a widely used mechanism in yeast that is likely to function in higher eukaryotes. We predict that metazoan SR-proteins with known functions in hnRNP formation, splicing and mRNA 3′end processing might also be able to affect elongation in a manner similar to Npl3.