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RNAs which guide RNA modification are found in eukaryotes and archaea, but not in bacteria, suggesting that this ancient site selection mechanism originated in the common ancestor of eukarya and archaea. In bacteria, the few RNA modifications (four methylations and 10 pseudouridylations in rRNA) are carried out using site-specific enzymes (Lafontaine & Tollervey, 1998; Gaspin et al., 2000; Ofengand et al., 2001; Bachellerie et al., 2002; Omer et al., 2003; Leppik et al., 2007; Ero et al., 2008). In archaea and eukaryotes where there is a need to modify more sites in rRNA, perhaps to fine-tune the efficiency of the ribosome, a different system for producing site-specific modifying enzymes evolved. This system, the snoRNPs, could use the same set of proteins for the modification of every target site just by changing the site recognition element. These recognition elements are now known as antisense elements in snoRNAs and sRNAs, respectively. This ancient mechanism appears to be successful as it has not changed a great deal as can be seen in the high conservation between archaea and eukarya guide sequences (Dennis et al., 2001; Omer et al., 2003; Dennis & Omer, 2005). To guide the tens or hundreds of modifications requires large sets of different snoRNAs/sRNAs which have evolved by duplications, mutations and selection of snoRNA/sRNA genes.

Generally, there are two modes of gene/gene cluster duplication: genes/gene clusters can be duplicated close to their origin (cis-duplication) or to a distant location, either on the same or a different chromosome (trans-duplication) (Figure 1.8). For instance, in vertebrates where the majority of snoRNA genes are single and intronic, cis- duplication has occurred when copies of the same intronic gene are found in different introns, neighboring ones or ones that are further away. Trans-duplication is detected when the same snoRNA gene is found within an intron of a different gene. Both duplication modes, although cis-duplication is far more frequent, might act on the same gene causing many widespread copies to be produced. For instance, in Platypus, paralogues of a box C/D snoRNA gene are found within the ribosomal protein S13 (RS13) gene as well as in a heat-shock protein 8 (Hsp8) gene and it has been concluded that a single trans-duplication occurred followed by further cis-duplication within one of the two genes (Figure 1.8) (Schmitz et al., 2008). It should be noted that it is crucial for

Chapter 1 small nucleolar RNA independent genes/gene clusters that their transcription elements are included in trans- duplication except when they are copied into existing gene clusters or introns. An example of trans-duplication followed by extensive cis-duplication (tandem repeats; see below) is seen in the HBII-52 cluster in humans. HBII-52 is a box C/D snoRNA which might regulate the alternative splicing of the serotonin receptor in human brains. It is thought that a snoRNA gene evolved in an intron of the SNRPB gene which was duplicated and gave rise to SNRPB including the snoRNA now called SNORD. SNORD is also a box C/D snoRNA which might guide methylation of the 28 rRNA. The snoRNA within the SNRPN, however, appears to be extensively duplicated resulting in 42 nearly identical copies (Yanget al., 2006; Nahkuriet al., 2008). In plants, where the majority of snoRNA genes occur in gene clusters, duplications are quite complex. Genes within a cluster, parts of a cluster or the whole cluster can be duplicated. Genes or gene clusters cis-duplicated adjacent to each other are called tandem repeats and are quite common and might be responsible for the origin of gene clusters (Brown et al., 2001; Brown et al., 2003a). A quite impressive case of tandem repeats is known from rice where a cluster (cluster 17) contains 42 genes which have arisen by five tandem repeats (Chen et al., 2003).

Figure 1.8: Cis- and transduplication of platypus box C/D box snoRNA paralogues.

Only one transduplication occurred followed by cis-duplication in one of the two genes. Filled ovals = platypus cDNA library snoRNAs; open ovals = snoRNAs found by blast search; hatched oval = non-functional in platypus but functional in human, mouse and cow. Figure is taken from (Schmitzet al., 2008).

Chapter 1 small nucleolar RNA At least 50% of the snoRNA genes in plants have two to four copies produced by the duplication modes described above. Furthermore, polyploidy is prevalent in plants and increases the allelic variants resulting in potential snoRNA redundancy. All in all, there are many genes and alleles producing snoRNAs which target the same nucleotide providing increased chances to accumulate mutations and, thus, generate new antisense sequences leading to new target sites which might be established under selection. For instance, twoArabidopsissnoR20 variants, located on two different chromosomes, target neighbouring sites of rRNA (Figure 1.9A). Mutations of an antisense element could also lead to the modification of a site that is distant from the original one as shown by the snoR16 variants (Figure 1.9B). These variants have two antisense elements, the one adjacent to the D’ box differs from the other variant by a few nucleotides and targets a completely different site (25S:Um2445 and 25S:Um36, respectively) (Brownet al., 2001;

Qu et al., 2001; Brown et al., 2003a). Thus, two or more snoRNA variants might have

diverged sufficiently (accumulation of nucleotide changes and indels) to become distinct snoRNAs. An example of the generation of novel snoRNA genes was provided by Brown et al. (2001). InArabidopsisthe double guide snoR15 gene was tandemly duplicated after transduplication to another chromosome. Due to mutation one snoR15 variant lost the function of the antisense element adjacent to the D box becoming the snoRNA gene U16, while the other variant lost the box D’ antisense element and became the snoRNA U55 gene (Figure 1.9C) (Brown et al., 2001; Brown et al., 2003a). It should be noted that mutations and selection could result in the loss of snoRNA function as well as leading to the production of non-functional pseudogenes which might be lost in time.

Chapter 1 small nucleolar RNA

Figure 1.9: Evolution of new snoRNA target sites and genes. A: the two snoR20 variants target the same rRNA but modify neighbouring sites. B: the two antisense elements adjacent to the D’ box of the two snoR16 variants differ in some nucleotides and base-pair with different rRNA sites. C: Transduplication followed by tandem duplication and loss of different antisense elements gave rise to U55 and U16 snoRNA genes (Barnecheet al., 2001; Quet al., 2001). m = methylation site. Figure is taken from (Brownet al., 2003a).

Chapter 1 small nucleolar RNA Other mechanisms to alter snoRNA gene diversity are gene conversion and unequal crossing over, which mostly lead to the loss of snoRNA genes and reorganisation of snoRNA gene clusters (Barneche et al., 2001; Brown et al., 2001; Qu et al., 2001). It is also feasible that due to unequal crossing over two different adjacent genes might fuse leading to a new chimeric snoRNA, as has be shown for ITS (see Alvarez & Wendel, 2003).

Comparisons of snoRNAs in different plant species have shown variation in the degree of conservation at the level of gene sequence and gene cluster organisation. For example, many orthologous genes show a high degree of conservation in their guide and box sequences while some paralogues appear to exhibit a high degree of variation. A high level of conservation indicates the action of purifying selection on the function of modifying specific target sites (Schmitzet al., 2008), whereas high sequence variation of paralogues might reflect active divergent evolution of snoRNAs. Not only are single snoRNA genes conserved between species, but the gene-order of some gene clusters is conserved as well. Furthermore, some gene clusters contain the same genes and sometimes other genes, which may be positioned in different orders in mixed clusters. In other species, however, these genes can be dispersed across the whole genome (dispersed cluster). For instance, the snoRNA gene cluster 7 in rice contains the same snoRNAs genes and gene order as does snoRNA gene cluster 21 inArabidopsis. However, while all of the cluster 7 genes can be found in rice intronic clusters 47 and 48, these clusters also contain another gene, snoR159, while cluster 47 also contains a copy of U18. These genes are located in Arabidopsis cluster 39 together with an inserted U54 gene (Figure 1.10). An example of dispersed cluster is shown in Figure 1.11. Some rice clusters (6, 15 and 17) have a 7 snoRNA gene core structure which in Arabidopsis is broken up into different clusters (40, 42 and 43) (Figure 1.11). The gene order of different clusters, either within the same or between different species, suggests that they are often subject to rearrangement in their evolution (Chen et al., 2003). An examination of snoRNA gene organisation in different species might provide insights into the reorganisation and transposition processes that occur during the evolution of different plant lineages (Brown

Chapter 1 small nucleolar RNA

Figure 1.10: Schematic illustration of conserved and rearranged gene order in rice

and Arabidopsis. Rice cluster 7 appears to be conserved because a similar cluster is

found in Arabidopsis(cluster 21). All cluster 7 genes are found in rice cluster 47 (mixed cluster) and Arabidopsis cluster 39, but either in a different order or with an additional gene inserted (U54). There is no U18 gene present in rice cluster 48. Figure is taken from (Chenet al., 2003).

Figure 1.11: Schematic illustration of dispersed gene clusters inArabidopsisrelative to rice.Rice clusters 6, 15 and 17 have a 7 snoRNA gene core structure which appears to have broken up into several clusters (40, 42 and 43) inArabidopsis. Some of the clusters found inArabidopsishave the same gene order (e.g. cluster 43 and 44) while others have a mixed (cluster 40) gene order compared to rice. While most of the clusters consist of heterologues (heterocluster), cluster 38 is a good example of a homocluster. Figure is

Chapter 1 small nucleolar RNA