RNAs that modify other RNAs

As we alluded to in the previous section, the primary function of some types of RNA is to catalyze modifications of other RNA sequences. In some cases, the RNAs are associated with proteins and form functional complexes, while in others the RNAs act in isolation.

Small nuclear RNAs

Small nuclear RNAs (snRNAs) are eukaryotic RNAs found in the nucleus that occur in conjunction with proteins as small ribonucleoprotein complexes (snRNPs). snRNPs are involved in a variety of nuclear regulatory processes, including intron splicing.

The spliceosome is composed of five snRNAs – U1, U2, U4, U5, and U6 – along with approximately 200 proteins (U3 is a snoRNA, see Section 2.3.2). Each intron consists of recognition sequences, located at the 5and 3splice sites where the flanking exons meet the intron, and a branch point site (BPS) located just upstream the 3

splice site. Assembly of the spliceosome begins at the 5splice site of the intron, where U1 snRNP binds the intron via base-pairing interactions. Next, the spliceosomal E complex is formed downstream the BPS, consisting of several RNA-binding proteins, which in turn recruit U2 snRNA and additional proteins to form the spliceosomal A complex. Next, a tri-snRNP consisting of U4/U6 snRNP and U5 snRNP subunits is recruited, which joins all of the components of the spliceosome together as the B complex. Conformational changes involving the release of U1 and U4 and the base pairing of U2 and U6 ultimately result in the catalytically active B* complex. The first splicing step occurs, in which cleavage at the 5 splice site occurs, resulting in a lariat structure formed between the 5 -most intronic nucleotide and the BPS nucleotide. At this point, the spliceosome exists as the C complex, further rearrangements occur, then the second catalytic step causes excision of the downstream exon and ligation with the already-freed upstream exon [26].

Some species contain an alternate spliceosome that is specific to a rare class of introns called U12 introns. This minor spliceosome is functionally analogous to the major spliceosome, except the corresponding snRNPs are U11, U12, U4atac/U6atac, and U5 (the only snRNP shared between the two spliceosomes).

Besides those involved in splicing, other snRNAs include the mammalian 7SK RNA, which along with HEXIM1 binds and negatively regulates the protein com- plex elongation factor P-TEFb, whose role is to activate RNA polymerase II; and telomerase RNA, the RNA component of telomerase, which maintains the length of telomeres, the protein-DNA structures that cap and protect the ends of chromosomes [27].

Small nucleolar RNAs

Sometimes classified as a subtype of snRNAs, small nucleolar RNAs (snRNAs) re- side in the nucleolus, the site of ribosome assembly, and as the RNA component of snoRNPs, help to catalyze the base modification of other RNAs in the nucleolus. Base modification is a post-transcriptional regulatory step and is often critical to the maturation of the RNA due to the subtle structural changes that result. There are a large number of annotated snoRNAs [28], which belong to one of two large sub- families based on the particular chemistry they catalyze. The C/D box snoRNAs catalyze methylation of the 2 oxygen on the ribose portion of the specific substrate nucleotide, while the H/ACA box snoRNAs catalyze pseudouridylation conversion of uridines – i.e., the isomerization of a normal uridine into a modified pseudouridine base. Each snoRNA has a specific substrate RNA that is determined by base pair- ing of the snoRNA sequence to its target; catalysis is carried out by the associated proteins in the snoRNP complex.

The majority of characterized snoRNAs have specificity to rRNAs and tRNAs, the major RNA species in the nucleolus. The pre-rRNA, for example, contains approxi- mately 200 modified bases, each catalyzed by a separate snoRNA [29]. All tRNAs also contain modified bases, a large number of which are created by snoRNAs. A subset of snoRNAs do not actually reside in the nucleolus; these small Cajal-body-specific RNAs (scaRNAs) guide the modification of the spliceosomal RNAs, which occurs in the Cajal body subnuclear organelles [30]. Still others (called “orphan” snoRNAs) have unknown targets, and may function on substrates not in the normal repertoire of snoRNAs [31].

RNase P and RNase MRP RNA

RNase P and RNase MRP are endoribonucleases, a class of enzymes that hydrolyze internal phosphodiester bonds in a ribonucleotide sequence, causing cleavage of an RNA strand into two pieces. Most endoribonucleases are composed exclusively of proteins; however, RNase P and RNase MRP are exceptions, consisting of both cat- alytic RNA and protein components. The RNA-induced silencing complex (RISC) is another exception (see Section 2.3.3).

RNase P is found throughout all lineages and in its primary capacity functions as a post-transcriptional modifier of tRNA molecules – tRNAs are transcribed with a leading 5 sequence that is removed upon maturation by the RNase P. RNase P might have a general role in the transcription and processing of several other Pol-III transcribed small RNAs including 5S rRNA, U6 snRNA, and 7SL RNA [32].

RNase MRP (mitochondrial RNA processing) is found only in eukaryotes and plays a role in mitochondrial DNA replication by cleaving the RNA primers used for DNA synthesis. It also has been shown in yeast to cleave the internal transcribed spacer 1 between 18S and 5.8S rRNAs in the large primary rRNA transcript [33, 34].

Autonomous ribozymes

A number of RNAs have distinct, independent catalytic ability and function in roles normally associated with protein enzymes. Accordingly, such RNAs have been called ribozymes (RNA enzymes). Technically any RNA with catalytic function can be con- sidered a ribozyme – 23S rRNA for example, despite residing in complex with several proteins, is in fact independently catalytic, and thus is a ribozyme [35]; similarly, RNase P and MRP are both ribozymes that are complexed with proteins.

prising the hammerhead, hairpin, Hepatitis delta virus (HDV), and Varkud satellite (VS) ribozymes. The best studied of these is the Hammerhead ribozyme, which is found in plant viruses and plays a role in viroid genome replication by trimming a newly generated RNA strand to the correct length [36, 37, 38]. The Hammerhead ribozyme is a self-cleaving RNA that consists of a three-stem-loop structure surround- ing an autocatalytic core sequence. The stems are numbered from 5 to 3 as I, II, and III according to the their position with respect to the site of cleavage, which occurs at an unpaired nucleotide upstream of the 3 strand of stem I. Hammerhead type I ribozymes are folded such that stem I is formed by the ends of the RNA sequence; type III is oriented such that stem III is formed by the end; type II is not known to exist in nature [39].

In vitro, the Hammerhead cleaving and target domains can be separated into

two different RNA molecules, such that the Hammerhead RNA can act in trans and catalyze cleavage of many RNA substrates. One notable application for such a system is the construction of molecule-level biosensors for the detection of specific nucleotide sequences [40].

Another class of ribozymes is the self-splicing introns. Similar to conventional introns, these sequences occur as spacer sequences between exons that are removed in a post-transcriptional modification step. However, the self-splicing introns do not use the canonical spliceosomal machinery to catalyze splicing. Group I introns are found in diverse transcripts and species and adopt a complex 10-hairpin (helices P1 through P10) structure, which contains a catalytic core. The 5 splice site first undergoes cleavage with a GTP cofactor, causing the upstream exon sequence to be covalently released from the intron, although it still remains associated with the intron through base pairing. Following a conformation change, cleavage and subsequent ligation with the 5 exon occurs at the 3 splice site, resulting in release of the intron and the ligated

exons as separate molecules [41].

Group II introns are found in the RNAs of organelles in protists, fungi, and plants; and also in bacteria. Their structure consists of six domains, dI - dVI, which con- tain the autocatalytic regions for splicing. In an analogous pathway to spliceosome- catalyzed splicing, the group II intron forms a lariat between a 3 bulged adenine nucleotide and the 5 splice site, followed by cleavage at the 3 splice site and ligation of the exon ends [42]. In vivo, this process is aided by additional protein factors [43], some of which are encoded in open-reading frames of the introns themselves. Ad- ditionally, several examples of nested introns, called twintrons, have been identified, such that an internal intron is spliced prior to the excision of the external intron [44].