The function of DHX15 and NKRF in ribosome biogenesis

Eukaryotic ribosome biogenesis is a highly complex process involving hundreds of assembly factors that are required for the sequential maturation of rRNA precursors and their association with ribosomal proteins. In human cells, three of the four mature rRNAs are co-transcribed into a single precursor, the 47S pre-rRNA transcript, in which the sequences of the 18S, 5.8S and 28S rRNAs are separated and flanked by spacer regions (5′ETS, ITS1, ITS2 and 3′ETS). Processing of this initial transcript to release the mature rRNAs takes place through a series of endonucleolytic cleavage events at specific sites in the spacer regions that are coupled in most cases with exonucleolytic digestion. The pathway of ribosome production is generally conserved across eukaryotes, but several characteristics, such as the presence of additional cleavage sites and the larger number of assembly factors, demonstrate the increased complexity of this process in human cells compared to yeast (Henras et al., 2015; Aubert et al., 2018). The function of most ribosome assembly factors remains to be determined.

In this study, novel interactions between the RNA helicase DHX15, the G-patch protein NKRF and the 5′-3′ exonuclease XRN2 were identified and the functions of these proteins in ribosome biogenesis were characterized. DHX15, NKRF and XRN2 were found to co-migrate with pre-ribosomal particles in sucrose gradients and to associate into a nucleolar subcomplex that is required for efficient pre-rRNA processing at site A′ in 5′ETS.

The A′ cleavage event is specific for metazoans and, although it generally takes place early

in the pre-rRNA maturation pathway, it was found to not be a pre-requisite for downstream processing and can also occur at later stages or be skipped altogether (Sloan et al., 2014).

This is consistent with our findings that depletion of these three factors does not affect the production of the mature ribosomal subunits despite the impaired A′ cleavage. Although the role of this additional processing event in metazoans is not known, the reduced cleavage at the A′ site upon knockdown of DHX15, NKRF and XRN2 leads to an accumulation of the 47S and 30SL5′ precursors. Interestingly, in MCF7 cells, the levels of 30SL5′ pre-rRNA are inherently higher than in other cell lines (Sloan et al., 2014). Furthermore, alternative processing pathways that generate different intermediates exist at several stages of pre-rRNA maturation and variations in the kinetics of these co-existing pathways that arise depending on the cell type or physiological conditions lead to changes in the ratios of the precursors. These different patterns of pre-rRNA processing were suggested to modulate the function of the ribosome, for example, by inducing distinct rRNA modification profiles depending on the precursors generated (Lafontaine, 2015; Aubert et al., 2018). Thus, even though the knockdown of DHX15, NKRF and XRN2 does not affect the production of the mature ribosomal subunits, it is possible that the increased levels of the 47S and 30SL5′

intermediates leads to subtle differences in rRNA modification that contribute to ribosome heterogeneity. This hypothesis is supported by the fact that 2′-O-methylation and pseudouridylation, which are the most abundant types of rRNA modification, occur at early stages of ribosome biogenesis, similar to A′ cleavage. Furthermore, sites with substoichiometric methylation levels as well as differences in the rRNA modification pattern between cell lines were recently discovered (Krogh et al., 2016).

The nucleolar interactions between DHX15, NKRF and XRN2 and the common A′

processing defect observed upon their knockdown strongly suggests that these proteins function together at this site. Consistent with this, our results showed that NKRF acts as a cofactor of DHX15 and stimulates its RNA binding affinity, ATPase and unwinding activities, and this catalytic activity of the helicase is necessary for proper A′ cleavage. On the other hand, the presence of XRN2 in this complex might serve as a quality control mechanism to enable the degradation of aberrant pre-rRNAs, similar to the function described for its mouse homologue (Wang and Pestov, 2011). The finding that the catalytic activity of DHX15 is required for efficient A′ cleavage suggests that the helicase might perform a structural remodeling event that facilitates processing, for example, by enabling the access of the currently unidentified endonuclease to its target site. This function would resemble the role proposed for its yeast homologue Prp43 in promoting the cleavage of 20S pre-rRNA by the endonuclease Nob1 (Pertschy et al., 2009). Alternatively, the action of DHX15 could lead

to the release or association of other proteins, such as factors that were previously implicated in A′ cleavage (Sloan et al., 2014).

The involvement of DHX15 in A′ cleavage represents the first function reported for this human helicase in ribosome biogenesis. As this processing step is specific for metazoans, this activity is not performed by its yeast homologue Prp43, for which other roles in this pathway have been described instead. Prp43 was suggested to participate in the biogenesis of both ribosomal subunits by promoting the final step of 18S rRNA maturation and mediating the association or release of snoRNAs during the assembly of the large ribosomal subunit (Bohnsack et al., 2009; Pertschy et al., 2009). These distinct activities of Prp43 in ribosome biogenesis are likely regulated by the G-patch cofactors Sqs1 and Pxr1 (Pertschy et al., 2009; Robert-Paganin et al., 2017). Thus, apart from its role in ensuring efficient A′ cleavage together with NKRF and XRN2, it is possible that DHX15 performs additional functions in this pathway with other G-patch cofactors, similar to Prp43.

Consistent with this, our results showed that the G-patch proteins GPATCH2, GPATCH4 and PINX1 stimulate the activity of DHX15 and are localized in nucleoli, implying that they might be involved in ribosome biogenesis. Since PINX1 was found to substitute the function of its yeast homologue Pxr1 in ribosome maturation, it likely performs a similar role in human cells (Chen et al., 2014; Robert-Paganin et al., 2017).

Apart from the common function of DHX15, NKRF and XRN2 in A′ cleavage, our data also revealed that knockdown of NKRF and XRN2 leads to the accumulation of several pre-rRNA fragments that are excised during processing and are normally targeted for degradation. The involvement of XRN2 in the turnover of these processing by-products has been previously described and our results further show that the similar defects induced by depletion of NKRF arise due to its role in recruiting the exonuclease to the nucleolus and to its pre-rRNA substrates (Wang and Pestov, 2011; Schillewaert et al., 2012; Sloan et al., 2013; Sloan et al., 2014). In addition, an increase in the levels of the 36S and 12S precursors was observed upon knockdown of NKRF and XRN2, which is probably a feedback effect caused by the failure to recycle the ribosome assembly factors bound to the excised pre-rRNA fragments. The common function of NKRF and XRN2 in pre-rRNA processing and turnover was also reported in a separate study, which found in addition that NKRF is upregulated during the heat shock response and restores nucleolar homeostasis by recruiting XRN2 to this subcellular location (Coccia et al., 2017).

Interestingly, it was shown that the function and localization of XRN2 are also modulated by the nucleoplasmic protein CARF, whose overexpression leads to an accumulation of XRN2 in the nucleoplasm and to similar pre-rRNA processing defects as those caused by depletion of NKRF or XRN2 (Sato et al., 2015). Similar to NKRF, CARF contains a

conserved domain that was suggested to mediate the interaction with XRN2 (XTBD; Richter et al., 2016). Although our results indicate that NKRF binds XRN2 in a different mode than CARF, taken together, these findings imply that the distribution of XRN2 between its nucleolar and nucleoplasmic functions is controlled by the interplay between NKRF and CARF. In contrast to its yeast homologue Rat1, whose exonuclease activity is stimulated by the cofactor Rai1, the regulation of XRN2 by XTBD-containing proteins does not seem to directly influence its enzymatic activity, which is probably due to the different binding mode compared to the yeast proteins (Miki et al., 2014; Sato et al., 2015; Richter et al., 2016).

Taken together, our results identify the G-patch protein NKRF as a key factor in ribosome biogenesis that mediates the assembly of a subcomplex containing DHX15 and XRN2 for facilitating A′ cleavage of the pre-rRNA transcript and also functions together with XRN2 in the turnover of pre-rRNA fragments excised during processing.