The predominant function of snoRNAs can be summarized as maturation of ribosomal RNA (rRNA) and small nuclear RNAs (snRNAs).
Ribosomal RNAs The ribosomes, are the macromolecules in the cells where the proteins are synthesized from messenger RNA (mRNA) templates. Beside many in- volved protein components, four RNAs are part of the two ribosome subunits. One, 5S ribosomal RNA (rRNA) is transcribed by RNA-PolIII, while the others are synthesized from a single rRNA operon by RNA-PolI. 18S, 5.8S, and 28S are separated through so called internal transcribed spacers (ITS) and framed by two external transcribed spac- ers (ETS) (Figure 2.5). A cascade of several endo- and exonucleolytic cleavage steps involving many trans-acting protein and snoRNA factors removes these transcribed spacers to produce the rRNAs. The interaction with snoRNAs SNORD3, SNORD14, SNORD22, SNORA63, SNORA73, and SNORD118 at specific sites is obligatory for correct cleavage steps (Henras et al., 2015).
Figure 2.5: Cleavage steps of the primary ribosomal RNA transcript and associated snoRNAs. Early maturation steps involve removal of ETS1, and cleavage of ITS1 to produce mature 18S rRNA, the RNA component of SSU. This requires snoRNAs SNORD3, SNORD14, SNORD22, SNORA73. The later cleavage steps produce mature 5.8S and 28S rRNA, the RNA components of LSU. Cleavages in ITS1, ITS2, and ETS2 require snoRNAs SNORD3, and SNORD118, SNORA63. Figure adapted from Bartschat (2011), Henras et al. (2015), and Maxwell and Fournier (1995).
Single Nucleotide Modifications More in the foreground of snoRNA research are the single nucleotide modifications within the rRNA sequences. They are introduced
in the nucleolus by the snoRNA machinery. For a proper function of ribosomes numer- ous 2’-O-methylations (currently 104 sites identified) and pseudouridylations (currently 96 sites identified) of single nucleotides in the RNA components of the ribosome are needed. While Pseudouridine (Ψ) is introduced by several pseudouridine synthase en- zymes (Pus) in bacteria, the task has been taken over by snoRNPs in archaea, protists, fungi, plants, and animals. Still Pus homologous that introduce Ψs in e.g. tRNAs (Spenkuch et al., 2014) are present in all organisms.
Pseudouridines The immediate effect of pseudouridines in the RNA sequence is an increased backbone rigidity through an additional hydrogen bond and improved base stacking (Spenkuch et al., 2014). Thus the modified bases stabilize secondary struc- tures and enables a certain degree of flexible conformation changes. Still only four Ψs during zebra fish development have been detected to have an individual distinct and finally lethal effect through brain malformation, organ, and/or body maldevelopment (Higa-Nakamine et al., 2012). Apart from that, prevention of a single pseudouridyla- tion in rRNAs has no measurable effect alone and only a disruption of several pseu- douridylations lead to decreased cell growth (King et al., 2003). As such the modified residues fine-tune and adapt the molecule structures also to dynamic environmental and developmental demands.
2’-O-methylation The effect of 2’-O-methylation of ribose is even less understood. On molecular level 2’-O-methylation is known to hinder alkaline degradation. Methylations at the 2-OH favors a special conformation of the ribose, block sugar edge interactions and change the hydration sphere around the oxygen (Helm, 2006). Thus, on a more general level it is thought to have impact on RNA structure. Recent high-throughput methods to identify methylation sites help to annotate methylations and subsequently understand their consequences (Birkedal et al., 2015; Krogh et al., 2016). Interest- ingly, the mentioned publication detected interdependence between distant modifica- tions. Furthermore, analysis of modification kinetics discovered that in LSU of yeast a subset of late post-transcriptional modifications can be distinguished from earlier co-transcriptional ones. All 2’-O-methylations in the SSU are of the latter class. Specialized Ribosomes We assume that snoRNAs play an important role in spe- cialization of the targeted RNA molecules to changing environmental conditions. In this scenario the modified residues adapt the ribosomes to the current cell challenges. Observed flexible snoRNA expression profiles, showing tissue and developmental depen- dent differential expression support this idea on the snoRNA side. On the modification
side evidence for specialized ribosomes can be drawn from some mTOR pathway in- duced Ψ residues that exist in the 28S rRNA of hamster ovary cells (Courtes et al., 2014). Also the possible between observed early and late 2’-O-methylations in the LSU, could reflect basic and adaptive rRNA modifications (Birkedal et al., 2015; Sloan et al., 2015). The concept of specialized ribosomes is not new, although the focus has rather been drawn on the interacting protein components so far (Xue and Barna, 2012). A specialized ribosome would be capable to favor the translation of a certain group of proteins.
Evolution of Modifications Interestingly, the general modification patterns of rRNAs (Appendix Figure A.1 and A.2) is retained during evolution making it even possible to identify distantly homologous snoRNAs based on their function (e.g. between yeast and human) (Ofengand and Bakin, 1997). In the rRNA molecules modification hotspots can be found in the regions that are also deeply conserved on sequence/structure level. The vast majority is located in the peptidyl transferase center and the intersubunit bridges, both highly functional and highly conserved regions of the ribosomes (King et al., 2003). Nevertheless, apart from the evolutionarily old and conserved modifica- tions, there are also hints for species specific ones. One documented example is the methylation of 28S-G3524 and 28S-C4004 (GENBANK X02995) detected in the rRNA of frog (X. tropicalis). The guiding snoRNAs NET1 and NET3 (non-eutherian spe- cific) are specifically found in Xenopus and some other vertebrate animals but neither in human nor any other placental mammals. This is in accordance with the detection of an unmethylated nucleotide at the analogue 28S rRNA site in human (Makarova and Kramerov, 2009), indicating a loss of function and the related gene.
Small Nuclear RNAs However, snoRNAs do not only target rRNAs, but were also discovered to guide modifications in small nuclear RNAs (snRNAs). The modified snRNAs constitute the RNA components of the spliceosomal machinery, that excises introns from primary RNAs transcripts (Section 2.1). The snRNAs (major and minor) are modified at a variety of sites. Responsible is a the Cajal body specific subset of snoRNAs: the scaRNAs. Accordingly, with the Cajal Body accumulation of snRNAs, also the scaRNA molecules amass in these cell organelles. Surprisingly, it became evi- dent that CB are not the site where snRNA modification is taking place, but snRNAs are also modified in the nucleolus. Yu et al. (2001) studied U2, the most extensively modified snRNA (ca. 10% of the nts undergo 2’-O-methylation or pseudouridylation) (Massenet et al., 1998) in Xenopus oocytes. Mutation of the U2s’ Sm-binding site
inhibits accumulation to the nucleolus, which correlates with unmodified U2 snRNA. Introduction of nucleolar localization signals (C-box and D-box) into the Sm-mutant U2 RNA re-establishes the modifications (Yu et al., 2001). As in rRNAs the modifi- cations within the snRNA sequences are conserved between species and are enriched in functional important regions, especially those of RNA-RNA contact (Karijolich and Yu, 2010) (Appendix Figure A.3). Interestingly, not only RNA-dependent, but also RNA-independent modifications occur. An example of such a site is Ψ at position U2- 35 in yeast and U2-34 in human. This site is exclusively catalyzed by Pus7p in yeast, while it is also established by the SCARNA8-snoRNP in human (Karijolich and Yu, 2010). In the snRNAs of the minor spliceosome fewer modifications, but at equivalent positions have been detected (Massenet and Branlant, 1999). It is suggested that the lesser extend of modification is correlated to an overall higher conversation of the minor introns (Karijolich and Yu, 2010).
Other Target RNAs Recent studies detected snoRNA guided pseudouridylation and 2’-O-methylation in further RNAs. SNORA70 and SNORA31 comprise antisense ele- ments (ASEs) for experimentally verified Ψs in RN7SK and RN7SL. Furthermore, mod- ified bases have been detected in snoRNAs themselves (Kishore et al., 2013) and in mR- NAs (Schwartz et al., 2014; Carlile et al., 2014). Newly developed high-throughput pro- tocols are capable of identifying base modifications in RNAs pulled from the cells. As snoRNAs and in consequence their editings are found differential expressed a broader variety of investigated samples from different tissues and under different environmen- tal influences will probably pinpoint more RNAs with snoRNA guided modifications, meaning that the interaction network is still increasing.