Despite the detailed knowledge of the individual protein components of H/ACA snoRNPs, little is known regarding the correlation between transcription and snoRNP assembly. In this study, we have found evidence of a cotranscriptional recruitment of some of the H/ACA snoRNP components and their involvement in the control of snoRNA biosynthesis.
It has been previously suggested that, in S. cerevisiae
, Naf1p and Shq1p play a key role in the metabolism of box H/ACA snoRNAs, aiding early steps of ribonucleoparticle assembly (6
). The yeast hnRNP-like protein Naf1p localizes to the nucleus, and even if it is not a stable component of the H/ACA snoRNPs, it is required for the accumulation of box H/ACA snoRNAs (6
). Moreover, Naf1p was shown to interact with the C-terminal domain of RNA Pol II and with the snoRNP core components Cbf5p and Nhp2p (8
In this article, ChIP experiments have shown that Naf1p is localized specifically on the coding region of H/ACA snoRNA transcription units while no interaction was observed on the promoter. This behavior, together with the evidence that Naf1p binds to RNA in vitro (8
), suggested that its recruitment could be triggered by the appearance of the appropriate RNA target sequences. In line with this hypothesis, an RNase treatment of the extract prior to immunoprecipitation produces a strong reduction of Naf1p association with H/ACA genes. Nevertheless, it cannot be excluded that Naf1p recruitment occurs already at the level of the promoter through the interaction with specific transcriptional factors and that it is subsequently mobilized to the CTD. In this case, this interaction could have been underestimated due to the detection sensitivity of the ChIP technique.
Chromatin immunoprecipitation analysis was carried out to monitor as well the recruitment of three H/ACA proteins, integral components of the mature snoRNP: Cbf5p, Nhp2p, and Gar1p. In analogy with Naf1p, Cbf5p and Nhp2p also show specific immunoprecipitation with the H/ACA genes and only with the transcribed regions. On the contrary, Gar1p does not show any significant reactivity with the H/ACA genes, suggesting that its association to the snoRNP occurs at later posttranscriptional phases. This is in agreement with previous results indicating that Gar1p is not essential for snoRNA accumulation, while it is required for the functionality of the mature particle (2
A biochemical investigation of the yeast proteome recognized a large number of factors physically linked to the phospho-CTD generated by the CTDK-1. These proteins are representative of a wide range of functions (e.g., transcription, RNA processing, chromatin structure, DNA metabolism, noncoding RNA biogenesis). The widespread nature of PCAPs points to a complex network of connections between Pol II elongation and other processes, expanding the role played by CTD phosphorylation in functional organization of the nucleus. Intriguingly, recent findings reveal a physical connection between phospho-CTD and protein factors having a function in snRNA modification and snoRNA biogenesis (8
). In line with this evidence, Cbf5p was copurified with the Spt5p elongation factor (31
) and Naf1p was shown to interact with the carboxy-terminal domain of the large subunit of RNA polymerase II (8
). The ChIP experiments performed in this work reveal that phosphorylation of serine 2 by Ctk1p contributes to the cotranscriptional recruitment of box H/ACA snoRNP proteins Naf1p and Cbf5p to the elongating RNA polymerase II, providing another link between snoRNP biogenesis and transcription elongation.
Recently, we showed that the methyltransferase Nop1p associates with box C/D snoRNA genes during transcription and, through its interaction with 3′-end termination machinery, provides a quality control for snoRNA synthesis profoundly influencing both transcription and 3′-end processing (33
). The cotranscriptional recruitment of Naf1p and Cbf5p proteins on the chromosome region of H/ACA genes makes them good candidates to control the 3′-end formation of the primary transcripts. As previously observed for C/D box snoRNAs, here we show that in a strain lacking the Cbf5p pseudouridylase, readthrough products extending to the downstream snoRNAs genes are detected. Thus, as for the C/D box, it is an integral component of the mature H/ACA particle that is the sensor controlling correct 3′-end formation.
Recent reports indicated that several subunits of the APT complex are required for 3′-end formation of snoRNAs (7
). In particular, Pti1p and Ref2p are required to prevent transcriptional readthrough into downstream genes and were suggested to function in uncoupling cleavage and polyadenylation (7
). The finding that Nop1p interacts with Ref2p and enhances its association with the C/D box snoRNA genes (33
) suggested a mechanism to mediate polyadenylation-independent 3′-end formation on the specific subpopulation of box C/D snoRNAs. Differently from the C/D box, no interaction was found between GST-Cbf5p, GST-Naf1p, and GST-Nhp2p fusion proteins and any of the APT (Ssu72p, Ref2p, Pti1p, Swd2p, Pta1p) and CFIA (Pcf11p, Clp1p, Rna14p, Rna15p) factors (data not shown) that were previously shown to be involved in snoRNA 3′-end formation (7
). Therefore, other still unidentified interactions are likely to mediate the observed coupling between snoRNP assembly and 3′-end formation of H/ACA snoRNAs.
In conclusion, we suggest that, also in the case of H/ACA snoRNA genes, a specific RNA factory is loaded on the nascent transcripts. Naf1p could be transferred during transcription elongation from the chromatin to the newly synthesize snoRNAs and in turn help the recruitment of Nhp2p and Cbf5p. Alternatively, Naf1p could interact with a preassembled complex as suggested by work of Henras and Wang (16
). In either case, the cotranscriptional recruitment allows the correct assembly of the H/ACA particle and, at the same time, provides a signal of quality control that allows correct and efficient 3′-end formation (Fig. ). Further support for the role played by Ser2 phosphorylation in this network is given by the finding that recruitment of Naf1p and Cbf5p on H/ACA snoRNA genes in ctk1
Δ cells is severely reduced. The strong readthrough phenotype observed on snoRNA genes in the absence of the Ctk1p kinase suggests that, besides recruiting H/ACA components, Ser2 phosphorylation brings in place 3′-end processing/termination factors, in analogy to what already shown for mRNA genes (1
). Future work will be required to address which ones are the factors of the 3′-end formation apparatus that are the sensors of successful H/ACA snoRNP assembly and to elucidate this complex network of interactions.
FIG. 5. The snoRNA factory. The snoRNP proteins are required for a quality control mechanism in which the functional assembly of box C/D and H/ACA snoRNAs is monitored during transcription. The snoRNA primary transcript attracts the 3′-end formation complex (more ...)