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RNA plays a central role in many cellular processes and several peculiarities of RNAs are probably relics of an ancient primordial RNA World. To fulfill their multiple present-day functions, these molecules need more than just four canonical bases. The numerous modified nucleosides that are formed during processing of nascent precursor RNA transcripts clearly serve this purpose. The recent discoveries of RNA-guided RNA modification machineries and of RNA editing processes leading to selected conversions of one base into another in the pre-RNA, add new dimensions to the problems surrounding the biosynthesis and functions of modified and edited nucleosides in RNA. The majority of these so-called minor or edited nucleosides appear to improve the performance of the matured RNA by working more efficiently and accurately in various steps of cellular metabolism. However, their effects can be subtle and not easy to demonstrate either or . Here, we review some basic characteristics of the modified nucleosides and of enzymes leading to such post-transcriptional modifications and editing of RNA

Post-transcriptional modifications at the first (wobble) position of the tRNA anticodon participate in the precise decoding of the genetic code that is mediated by the codon-anticodon interaction. However, the biosynthesis and functions of many wobble modifications remain unknown. We describe, here, a reverse genetic approach that we used to explore the uncharacterized genes of and yeast that are responsible for the wobble modifications (the Ribonucleome analysis). By combining this method with a comparative genomics approach, we identified an essential gene () that is responsible for the biosynthesis of lysidine at the wobble position of the bacterial tRNA that is specific for the AUA codon. Lysidine is an essential wobble modification that is required for the identity of the tRNA and its AUA codon specificity. reconstitution of the wobble modification revealed the detailed mechanism by which lysidine is synthesized.

Accurate maintenance of wobble modifications is, thus, required for various biological functions. We also show that the subcellular localization of tRNAs in is controlled by different wobble modifications. Moreover, we describe our recent studies that have revealed that the lack of wobble modification of mitochondrial tRNAs leads to translational defects that are associated with mitochondrial diseases, which suggests that disordered RNA modification may be a causative factor of human diseases.

Trypanosomatids include a number of protozoan parasites that infect over 27 million people worldwide. Besides their medical importance, these organisms have also provided a wealth of novel biological discoveries including: RNA editing, mRNA trans-splicing, eukaryotic poly-cistronic transcription, and a mechanism for large-scale mitochondrial tRNA import. For many years, the study of RNA post-transcriptional modification in trypanosomatids has lagged behind when compared to bacterial, yeast, and animal systems. However, the discovery of editing in tRNA and 7SL RNAs has produced renewed interest in the processing of non-coding RNAs in these organisms. This chapter will compile what is currently known about RNA editing and modification in trypanosomatids, emphasizing the role these processes play in the structural reshaping of non-coding RNAs. Due to a number of substantive recent reviews, mRNA editing will not be the subject of this chapter. In addition, snoRNA-mediated modification of ribosomal RNAs will be covered in chapter 8 of this book.

Transfer RNAs are adaptor molecules, which decode mRNA into protein and, thereby, play a central role in gene expression. During the maturation of a primary tRNA transcript, specific subsets of the four normal nucleosides adenosine, cytidine, guanosine, and uridine are modified. The formation of a modified nucleoside can require more than one gene product and may involve several enzymatic steps. In the last few years, the identification of gene products required for formation of modified nucleosides in tRNA has dramatically increased. In this review, proteins involved in modification of cytoplasmic tRNAs in are described, emphasizing phenotypic characteristics of modification deficient strains and genetic approaches used to determine the in vivo role of modified nucleosides/modifying enzymes.

Determining the function of single nucleotide modifications in tRNA has been elusive because so many tRNA modification enzymes are not essential for cell viability, making it difficult to do functional studies in vivo. The enzyme that catalyzes the formation of 1-methyladenosine modification at position 58 (mA58) in most yeast tRNAs is essential for yeast cell viability, which has made it possible to explore the role of this single modification in tRNA structure and function. In addition to reviewing the role of mA in tRNAs from prokaryotes to eukaryotes and mitochondria to cytoplasm, this chapter discusses the importance of mA58 in maintaining the 3-dimensional structure of yeast initiator tRNA. Exploiting the genetics available in yeast, it has been discovered that initiator tRNA lacking mA58 is eliminated from cells by 3’ polyadenylation and 3’ to 5’ exonuclease degradation.

Modified nucleosides are present in mRNA of all eukaryotes, albeit at much lower levels than in other RNA moieties such as rRNA, tRNA, and snRNA. Modification by methylation occurs on the terminal guanosine of the cap (N-methylguanosine), and the first two encoded nucleosides (2’-O-methylnuculeosides) in most higher eukaryotes. Additional modifications of cap nucleosides occur in special cases where the cap is derived by transsplicing in nematodes and kinetoplastids. Modification by methylation also occurs at internal adenosine residues in many species (N-methyladenosine). Modification by deamination occurs at specific adenosine residues (forming inosine) and cytidine residues (forming uridine) in very specific cases leading to post-transcriptional editing. Numerous studies have shown the importance of the cap N-methylguanosine in translation, splicing, transport, and mRNA stability. The role of the 2’-O-methylnucleosides is not as well understood, but there is evidence that these modifications play some role in translation efficiency. The role of internal N-methyladenosine residues is least known, and is the focus of this review. The formation of N-methyladenosine is catalyzed by a complex enzyme containing a subunit (MT-A70) that co-localizes with nuclear speckles and appears to be widely expressed in all higher eukaryotes. Loss of this enzyme leads to a sporulation defect in yeast and to apoptosis in mammalian cells, although the exact mechanism by which the effects occur remains obscure.

The biogenesis of spliceosomal UsnRNPs in higher eukaryotes involves a nucleocytoplasmic shuttling cycle. After transcription and processing in the nucleus, the mG-cap-dependent export of the snRNAs U1, U2, U4, and U5 to the cytoplasm occurs. In the cytoplasm, these UsnRNAs specifically associate with seven Sm-proteins and form a doughnut-shaped snRNP core structure. This assembly, mediated by the SMN complex, is a prerequisite for the hypermethylation of the mG-cap to the 2,2,7-trimethylguanosine (mG)-cap. Snurportin1 (SPN1), specifically, recognises the m3G-cap and facilitates the nuclear import of UsnRNPs. The recently determined crystal structure of human SPN1 reveals a significantly different binding mode for the cap structure in comparison to that of the m7G-binding proteins CBC, eIF4E and VP39.

A pseudouridine () residue in a phylogenetically conserved position of U2 snRNA that pairs with the intron to form the pre-mRNA branch site helix of has been shown to induce a dramatically altered architectural landscape compared with that of its unmodified counterpart. In the y-dependent structure the branch site adenosine in an extrahelical position, with the nucleophilic 2’OH positioned at the surface of the widened major groove. Clustering of electronegative functional groups and kinking of the backbone in the modified structure also result in a region of exceptional negativity in the region of the 2’OH. These features may assist in recognition and activity of the branch site during the first step of splicing. This is the first case in which a native y has been shown to induce a major alteration in structure. However, it is likely that other conserved modification sites in the spliceosome and ribosome may impact structurally on assembly and function.

RNA-guided 2’--methylations and pseudouridylations occur in several different types of RNAs and in a wide range of organisms. Hundreds of the RNAs that guide these modifications have been identified, leading to breakthroughs in our understanding of the mechanisms of RNA-guided RNA modifications and, to some extent, the functions of 2’--methylated residues and pseudouridines. There are two classes of guide RNAs, namely box C/D and box H/ACA RNAs, which direct 2’--methylations and pseudouridylations, respectively. The guide RNAs function primarily by binding to complementary regions in the target RNAs. Cel-lular guide RNAs exist in RNA-protein complexes comprised of one guide RNA and a set of proteins that includes the modifying enzyme (2’--methylase or pseu-douridylase). We are beginning to understand the basis for the importance of the RNA-guided modifications, which are well conserved and clustered in function-ally important regions of RNAs. Recent reports indicate that modified nucleotides in rRNAs and spliceosomal snRNAs contribute to protein synthesis and pre-mRNA splicing, respectively.

rRNA maturation requires extensive covalent modifications of riboses and bases. These modifications concern exclusively the most conserved regions of the molecule, and some modifications are highly conserved throughout the evolution. In bacteria, rRNA modification is achieved exclusively by site-specific enzymes while in archaea and eukaryotes the formation of 2’--methylriboses and pseudouridines is guided by numerous snoRNA that direct a catalytic machinery to the target sites on the pre-rRNA. The exact function of these modifications remains elusive since preventing their formation generally leads to no detectable phenotype. However, most of the enzymes that catalyze the formation of these modifications are encoded by essential genes in yeast. Moreover, in some cases preventing the formation of several modifications simultaneously affect ribosome biogenesis and translation. This review presents rRNA modifications that have been conserved throughout the evolution and it gives a special emphasis to the recently characterized 2’--ribose RNA methyltransferase Spb1p, which broke the “snoRNA-guided only” methylation dogma.