Alteration of 28S rRNA 2'-O-methylation by etoposide correlates with decreased SMN phosphorylation and reduced Drosha levels.

The most common types of modification in human rRNA are pseudouridylation and 2'-O ribose methylation. These modifications are performed by small nucleolar ribonucleoproteins (snoRNPs) which contain a guide RNA (snoRNA) that base pairs at specific sites within the rRNA to direct the modification. rRNA modifications can vary, generating ribosome heterogeneity. One possible method that can be used to regulate rRNA modifications is by controlling snoRNP activity. RNA fragments derived from some small Cajal body-specific RNAs (scaRNA 2, 9 and 17) may influence snoRNP activity. Most scaRNAs accumulate in the Cajal body - a subnuclear domain - where they participate in the biogenesis of small nuclear RNPs, but scaRNA 2, 9 and 17 generate nucleolus-enriched fragments of unclear function, and we hypothesize that these fragments form regulatory RNPs that impact snoRNP activity and modulate rRNA modifications. Our previous work has shown that SMN, Drosha and various stresses, including etoposide treatment, may alter regulatory RNP formation. Here we demonstrate that etoposide treatment decreases the phosphorylation of SMN, reduces Drosha levels and increases the 2'-O-methylation of two sites within 28S rRNA. These findings further support a role for SMN and Drosha in regulating rRNA modification, possibly by affecting snoRNP or regulatory RNP activity.

Altered dynamics of scaRNA2 and scaRNA9 in response to stress correlates with disrupted nuclear organization.

Small Cajal body-specific RNAs (scaRNAs) are part of small Cajal body-specific ribonucleoproteins (scaRNPs) that modify small nuclear RNA (snRNA) in Cajal bodies (CBs). Several scaRNAs (scaRNA 2, 9 and 17) have been found to generate smaller, nucleolus-enriched fragments. We hypothesize that the fragments derived from scaRNA 2, 9 and 17 form regulatory RNPs that influence the level of modifications within rRNA by altering small nucleolar RNP (snoRNP) activity. Here we show that external factors such as DNA damaging agents can alter the scaRNA9 full length to processed fragment ratio. We also show that full-length scaRNA2 levels are likewise impacted by DNA damage, which correlates with the disruption of SMN, coilin and WRAP53 co-localization in CBs. The dynamics of scaRNA9 were also shown to be affected by Drosha levels, which suggests that this protein may participate in the biogenesis and processing of this non-coding RNA. Identification of factors that contribute to scaRNA 2, 9 and 17 processing may facilitate an assessment of how external stress can lead to changes in rRNA modifications.

Identification of additional regulatory RNPs that impact rRNA and U6 snRNA methylation.

Ribosomes can be heterogeneous, and the major contributor to ribosome heterogeneity is variation in rRNA modification. There are two major types of rRNA modification, pseudouridylation and ribose methylation. In humans, the majority of these rRNA modifications are conducted by two classes of small nucleolar ribonucleoproteins (snoRNPs), which contain a guide RNA (small nucleolar RNA, snoRNA) complexed with proteins. Box H/ACA snoRNPs conduct pseudouridylation modifications and box C/D snoRNPs generate ribose methylation modifications. It is unclear how ribosome heterogeneity is accomplished in regards to the understanding of the signals and factors that regulate rRNA modifications. We have recently reported that a new class of RNP, that we term regulatory RNP (regRNP), may contribute to rRNA modification as well as the modification of nucleolar trafficked U6 snRNA, via interactions with snoRNPs. Here we report the identification of additional regRNP activities that influence the methylation of two sites within 18S rRNA, two sites within 28S rRNA and one site within U6 snRNA. These findings provide additional proof that regulation of snoRNP activity contributes to ribosome heterogeneity.