RNA Processing

Pre-rRNA processing analyses revealed only minor delays in the maturation of the 18S and 25S rRNA precursors, and the most important contribution to the decreased steady-state levels of rRNAs is an increased instability, probably caused by partial degradation of functionally impaired ribosomes [150].

From: The Enzymes , 2017

Well-Known Combined Immune Deficiency Syndromes

John B. Ziegler , Sara Kashef , in Stiehm's Immune Deficiencies, 2014

Pathogenesis

RMRP is mainly located in the nucleolus in which it participates in generation of ribosomes by performing the endonucleolytic cleavage of the rRNA that ultimately leads to generation of mature 5.8S rRNA (Figure 6.4A). RMRP also processes mitochondrial RNA primers (an essential step to activate mitochondrial DNA replication) and participates in cell cycle control by cleaving cyclin mRNA (Figure 6.4B). 100 This is in accord with the generalized defect in cell growth observed in T cells, B cells, and fibroblasts, 103–105 and could thus explain many of the features of CHH. Recently, an additional function was established by the observation of the interaction of RMRP with the human telomerase catalytic subunit (hTERT). 106 Interestingly, the 3′ end of RMRP is essential for the formation and the activity of the hTERT–RMRP complex exhibiting RNA-dependent RNA polymerase activity. The hTERT–RMRP complex negatively regulates RMRP levels, but may also have an effect on other genes yet to be characterized. 107

Figure 6.4. Known function of the RNase MRP complex. (A) In human rRNA, cleavage at the upstream 5.8S rRNA junction site is necessary for proper ribosome assembly and is associated with the degree of bone dysplasia, (B) whereas mRNA cleavage of cyclin B2 mRNA is necessary for cell cycle progression and is associated with the additional features like susceptibility to cancer, immune deficiency, anemia, and hair hypoplasia. (C) At least the RMRP ortholog in yeast is involved in the processing of mitochondrial RNA, which functions as primer for mitochondrial DNA replication. (D) Recently a new interaction with the human telomerase catalytic subunit (hTERT) revealed an RNA-dependent RNA polymerase activity leading to siRNA altering gene expression.

 Figure reproduced from Thiel and Rauch, 107 with permission.

In CHH, in vitro lymphocyte proliferation is impaired and reduced secretion of IL-2 and IFN-γ and defective expression of IL-2 receptor α have been found. The proliferative defect cannot be rescued either by addition of exogenous IL-2 or by stimulation with phorbol myristate acetate and ionomycin – agents that bypass receptor-mediated signaling. CHH patients have a reduced number of naïve (CD45RA+) T lymphocytes. There appears to be increased apoptosis of circulating T lymphocytes associated with increased expression of Fas and Fas ligand (FasL) and of the proapoptotic molecule Bax, whereas expressions of the antiapoptotic molecules bcl-2 and inhibitory of apoptosis (IAP) are reduced. As mentioned above, RMRP immune deficiency-causing mutations compromise cyclin B2 mRNA cleavage and may therefore result in increased levels of cyclin B2. Overexpression of this molecule leads to accumulation of cells in late mitosis and contributes to chromosomal instability. 100

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Messenger RNA Processing in Eukaryotes

K. Potter , ... J.A. Wise , in Encyclopedia of Biological Chemistry (Second Edition), 2013

Abstract

RNA processing is the term collectively used to describe the sequence of events through which the primary transcript from a gene acquires its mature form. Very soon after synthesis by RNA polymerase II begins, transcripts from nuclear protein-coding genes acquire a 5′ cap structure. The 3′ end of the messenger RNA (mRNA) is modified by the addition of a long string of adenosines in a process tightly linked to transcription termination. Finally, maturation of most eukaryotic mRNA precursors requires a process known as splicing, in which internal noncoding segments known as introns are removed and the coding segments, known as exons, are joined to produce functional mRNAs. In complex eukaryotes, exons are much smaller than introns and provide the functional unit initially recognized by the splicing machinery. Because the splicing signals found at the exon/intron boundaries have low information content, ancillary elements known as splicing enhancers and silencers are required to specify the precise sites of exon joining. Throughout their life cycles, mRNAs are decorated with proteins, some stably bound and others associated only transiently; thus, the functional form recognized by both the RNA-processing machinery and the translating ribosome is a ribonucleoprotein complex (mRNP). An important advance in recent years is the discovery that RNA-processing events are mechanistically coupled to transcription by RNA polymerase II.

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Ribonucleases - Part B

Clare Simpson , David Stern , in Methods in Enzymology, 2001

A Role of RNA Processing and Decay in Regulating Chloroplast Gene Expression

RNA processing in chloroplasts includes mRNA 5′- and 3′-end processing, intron splicing, and intercistronic cleavages of polycistronic messages, as well as typical tRNA and rRNA processing. These posttranscriptional steps, along with changes in RNA stability, have received considerable attention for two reasons. First, changes in chloroplast gene expression during chloroplast biogenesis or in response to environmental signals have much more often been shown to be posttranscriptional rather than transcriptional. Second, genetic studies designed to identify nuclear mutants defective in chloroplast function have nearly exclusively identified posttranscriptional defects. These data, along with in vitro systems developed to dissect the relevant mechanisms, have been the subject of a number of reviews. 1−6

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mRNA 3' End Processing and Metabolism

Hari Krishna Yalamanchili , ... Zhandong Liu , in Methods in Enzymology, 2021

4.7.1 Integration with CLIP-seq data

RNA processing readouts like alternative splicing and alternative polyadenylation can be substantiated by matching CLIP-seq ( Stork & Zheng, 2016) signal near the splice sites or cleavage sites (Zhu et al., 2018), respectively. For demonstration purpose here we used publicly available NUDT21 CLIP-seq data in GEO database (GSM917661). Processing of CLIP-seq data is described elsewhere (Uhl, Houwaart, Corrado, Wright, & Backofen, 2017). Fig. 6A and B illustrate 3′UTR shrinking in PAK1 and VMA21, respectively. Corresponding NUDT21 binding (CLIP-seq signal) in the proximity of the polyadenylation sites are marked with arrow heads. CLIP-seq signal substantiate the 3′UTR shrinking of PAK1 and VMA21 as the direct consequence of NUDT21 KD.

Fig. 6

Fig. 6. Integration with CLIP-seq data: CLIP signal overlapping with PAC-seq signal for (A) PAK1 and (B) VMA21.

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Genomics in Multiple Myeloma

Francesca Cottini , ... Giovanni Tonon , in Cancer Genomics, 2014

Mutations in RNA Processing Genes

RNA processing is a brand new field in cancer biology, starting with the identification of aberrancies in microRNA production and, more recently, highlighted by the discovery of mutations involving multiple components of the RNA splicing machinery such as U2AF35, ZRSR2, SRSF2 and SF3B1 in myelodysplastic syndromes and other cancers [73].

DIS3 is mutated in 10–18% of MM [65,74]. It encodes a highly conserved RNA exonuclease, which serves as the catalytic component of the exosome complex involved in regulation of processing and abundance of different RNA species. The four observed mutations occur at highly conserved regions and cluster within the RNA domain facing the enzyme's catalytic pocket. Thus, DIS3 mutations might deregulate protein translation and mRNA processing. Even though the precise role of DIS3 in MM pathogenesis is still unknown, patients whose MM bears DIS3 mutations often exhibit deletion of the remaining DIS3 allele, suggesting that these mutations can complete the loss of function of the protein. However, other evidence suggests that one of the mutations can represent a gain of function. Another interesting finding is that DIS3 mutations are associated with deletions of the RB1 region, as reported recently [74]. Whether these events are collaborative and may elicit a synergic effect in MM growth and progression is still unknown. In addition, mutation of DIS3 may be enriched in MM with either a t(4;14) or t(11;14) [74].

FAM46C gene is mutated in 13% of patients, and its genomic locus is deleted in 10–15% of cases. FAM46C deletion is associated with a worse prognosis [46]. It belongs to the Ntase fold protein superfamily, which transfers NMP from NTP to an acceptor hydroxyl group of protein or nucleic acid. FAM46C function is still unknown, but GSEA algorithms define a concomitant expression of FMA46C with a set of ribosomal proteins known to be tightly co-regulated and involved in initiation and elongation of protein translation. Similarly, FAM46C can also behave as an mRNA stability factor [46,65].

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Ribonucleases - Part B

Karsten Liere , ... Gerhard Link , in Methods in Enzymology, 2001

In Vitro Redox Assays of p54

RNA-binding and processing assays are carried out in standard reaction mixtures containing p54 (see above). They are pretreated, however, with a 20   mM concentration of oxidant menadione (K3; Sigma), cystine (CysCys; Sigma), or oxidized glutathione (GSSG; Sigma), or with reductant 2-mercaptoethanol (EtSH; Sigma), dithiothreitol (DTT; Sigma), cysteine (CysSH; Sigma), or reduced glutathione (GSH; Sigma). The pretreatments are at room temperature for 10   min before the addition of labeled RNA. Stock solutions (100   mM) of the redox reagents are prepared in 50   mM Tris-HCl, pH   8.0. Escherichia coli thioredoxin (5 μM; BRL, Gaithersburg, MD) is reduced with a 5000 M excess of DTT or is oxidized with a 5000-fold excess of menadione before use.

To confirm that p54 processing activity is specifically modulated by glutathione, redox reversibility assays are carried out. Purified p54 is preincubated with 20   mM oxidized or reduced glutathione, menadione, or DTT for 5   min and is subsequently treated with equimolar amounts of the indicated redox reagent for an additional 5   min (Fig. 2B).

To test the extent to which phosphorylation and redox state act together in the control of p54 activity in vitro, processing experiments are carried out with p54 that has been pretreated in various combinations (Fig. 2C). In experiments involving initial treatment with the kinase or CIAP, followed by the redox-reactive reagent, the two steps are separated by reisolation of the protein as described above. In contrast, when p54 is first treated by redox reagents and then subjected to phosphorylation or dephosphorylation, the latter treatments can be carried out without prior reisolation of p54. In the case of phosphorylation, after preincubation with the respective redox reagent, a mixture is added containing 2 μl of 10   ×   kinase buffer, 0.2 μl of 20   nM ATP, and 0.5 μg of PKA (20-μl final reaction volume; containing 10   ×   kinase buffer, 0.2   mM ATP) and the sample is incubated at 30° for 30   min. Similarly, dephosphorylation after redox treatment is done by adding a mixture of 2 μl of 10   ×   CIAP buffer and 20   units of CIAP in a final reaction volume of 20 μl, and incubation of the sample is continued at 30° for 30   min. To start the subsequent RNA-binding or processing reactions, radioactively labeled RNA is added and the samples are incubated at room temperature according to the protocols given above.

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Rhabdovirus

Wang-Shick Ryu , in Molecular Virology of Human Pathogenic Viruses, 2017

14.4 Effects on Host

CPE: VSV-infected cells are lysed and as a result, plaques are formed. How does VSV infection lead to cell lysis? VSV infection blocks host cell protein synthesis, while viral protein synthesis remains unaffected. Two mechanisms for "host shutoff" function have been revealed, as shown below.

RNA Processing : First, VSV infection blocks the nuclear export of cellular pre-mRNA. Specifically, it was shown that viral M protein binds to Rae1, 9 a nuclear export factor, thereby blocking the nuclear export of cellular pre-mRNA (Fig. 14.7). It should be noted that this inhibitory mechanism does not affect the viral mRNA synthesis, as the viral mRNA synthesis is limited to the cytoplasm. Hence, this mechanism is also dubbed "host shutoff."

Figure 14.7. Host shutoff functions by VSV M protein.

Cellular pre-mRNAs are exported to cytoplasm via nuclear pore. TAP/p15 complex serves as a transport receptor for pre-mRNAs, and recruits pre-mRNAs to NPC (nuclear pore complex). VSV M protein blocks the nuclear export of pre-mRNAs via its interaction with Rae1 molecule, a nuclear export factor. Note that Rae1 acts as a nuclear export factor via its interaction with Nup98, a component of NPC. In addition, VSV inhibits cellular translation via two mechanisms: (1) by enhancing eIF2 phosphorylation and (2) by suppressing eIF4E phosphorylation.

Translation: In addition, VSV infection blocks cellular translation. First, the phosphorylation of eIF4E 10 is reduced in VSV-infected cells, thereby reducing eIF4F 11 complex formation. On the other hand, the phosphorylation of eIF2 is increased in VSV-infected cells, thereby decreasing translation. Perhaps, the phosphorylation of eIF2 12 by IFN-activated PKR greatly contributes to the translation suppression (see Fig. 5.8). An intriguing point is that VSV protein translation remains unaffected and the viral proteins accumulated, although host translation function is substantially impaired. The question of how selectively VSV mRNA translation is unaffected remains unknown.

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Epigenetic Shaping of Sociosexual Interactions

Ryohei Sekido , in Advances in Genetics, 2014

4.3 SRY May Regulate Pre-mRNA Splicing

RNA processing is also important to elicit different biological function from an RNA transcript. Several lines of evidence demonstrate that RNA processing is crucial for sex determination. For example, in Drosophila, a ratio of X chromosomes to autosomes affects the expression of sex-lethal (Sxl) gene that encodes an RNA-binding protein. In females, a high level of SXL protein promotes an alternative splicing of transformer (tra) pre-mRNA to be translated into the functional TRAF protein, which is also an RNA-binding protein. TRAF in turn promotes another alternative splicing event to produce doublesex (dsx) mRNA for the female-specific form of DSX protein (DSXF) and fruitless mRNA that encodes no protein, while the dsx and fru genes produce the male-specific mRNAs for DSXM and FRUM proteins in the absence of TRAF, respectively. The production of the correct form of DSX and the presence or absence of FRU in each sex controls sexual differentiation and sexual behavior. DSX controls sexual differentiation, including pheromone production, as well as sexual behavior (Rideout, Dornan, Neville, Eadie, & Goodwin, 2010). FRU is a chromatin-associated protein required for the development of male courtship circuits (Demir & Dickson, 2005; Ito et al., 2012; Kimura, Ote, Tazawa, & Yamamoto, 2005; Manoli et al., 2005).

In some cell lines, SRY protein colocalizes with β-catenin, splicing factor U2AF65, and RPS7 and RPL13a in nuclear speckles (Bernard et al., 2008; Ohe et al., 2002; Sato et al., 2011). It is known that ribosomal proteins are imported into the nucleus and assembled with ribosomal RNA to form spliceosomes. These suggest that SRY plays a role in alternative pre-mRNA splicing, and indeed SRY facilitates alternative splicing of several well-known pre-mRNA substrates in an in vitro assay (Ohe et al., 2002). It is unknown how SRY localizes in nuclear speckles. The evidence that β-catenin changes its subcellular localization by posttranscriptional modifications (Brembeck et al., 2004) and physically interacts with SRY (Bernard et al., 2008), suggests that β-catenin recruits SRY to nuclear speckles. Moreover, an X-linked orphan nuclear receptor, Dax1, also acts as a shuttling RNA-binding protein between the nucleus and the cytoplasm (Lalli, Ohe, Hindelang, & Sassone-Corsi, 2000). Dax1 has been thought to be antagonistic to Sry activity in sex determination because its overexpression causes XY sex reversal in the presence of Sry (Swain, Narvaez, Burgoyne, Camerino, & Lovell-Badge, 1998), although the molecular basis of this antagonism remains to be explored. Recent studies have demonstrated that Dax1 also interacts with U2AF65 (Ohe, Tamai, Parvinen, & Sassone-Corsi, 2009), suggesting that Dax1 might inhibit SRY-mediated pre-mRNA splicing by competing with U2AF65.

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RNA and aging

Milan Mušo , in Rna-Based Regulation in Human Health and Disease, 2020

Discussion and outlook

Multiple RNA processing mechanisms are altered in aging. Dysregulated RNA splicing, downregulation of RNA silencing, NMD, upregulation of circRNAs and prolonged 3′UTRs in senescent cells could expand the aging hallmarks and serve as new aging biomarkers ( Fig. 15.3). Experiments in model organisms showed a key role of RNA splicing and RNA silencing in regulating lifespan through interaction with established longevity pathways such as the IIS and stress response. In aging humans, RNA processing is also deregulated, but its direct significance to the control of human lifespan remains to be tested. Interestingly, changes in RNA splicing might affect different pathways to those affected by age-associated expression changes, suggesting that transcriptional and post-transcriptional mechanisms could contribute to different aspects of aging. On the other hand, RNA processing events were shown to be intertwined with transcription, chromatin regulation and also with each other. Thus to determine the temporal hierarchy and contributions of different processes to aging in cells, systems biology approaches will be required to analyze the aging chromatin, transcriptome and proteome simultaneously, ideally on a single cell level. One such method, single-cell nucleosome, methylation and transcription sequencing (scNMT-Seq) analyses chromatin accessibility, DNA methylation and gene expression in a single cell and could thus answer some of the above-mentioned questions [129]. scRNA-seq will likely continue to be at the center of future revelations about the aging transcriptome and new methods such as RNA-editing using Cas13 might prove helpful in testing the specific effects of different RNA species on aging [130]. This is important, because not all age-associated transcriptomic signatures are detrimental. For example, downregulation of ribosome components with age is most likely a protective response to age-linked pathology [48]. Similarly, RNAi silencing might contribute to aging only through alg-1, but not alg-2 [100]. Thus, delineating aging-promoting effects from protective responses will be crucial for identifying potential targets for therapies. Finally, according to the omnigenic theory, almost every biological process will have at least a minute contribution to complex phenotypes [131]. Lifespan and aging are perhaps some of the most complex phenotypes and although conserved core pathways exist, it is likely that to dramatically slow down aging, alternations of multiple pathways, such as through multiplexed genetic engineering, will eventually be required.

RNA-based regulation is especially important for neuronal tissues which are enriched for RNA splicing, lncRNAs and age-linked accumulation of circRNAs [77,122,132]. Similarly, lifespan-extension in C. elegans through alternations of the NMD pathway is dependent on neurons [88] The long-lived and post-mitotic nature of these cells might make these cells more reliant on tight RNA quality control. It is likely that RNA has further still undiscovered roles in neurones. For example, it was shown that both short and long RNAs might regulate phase-separation to form cytoplasmic stress granules and protein aggregates [133]. It will be interesting to see whether transcriptomic and post-transcriptomic changes contribute to the protein accumulation seen in neurodegeneration and aging brains.

Aging research is in burning need for clinical biomarkers to facilitate effective genetic studies of human longevity and personalized medicine approaches. circRNAs are the best correlated class of RNA with age [77]. Similarly, splicing of genes might be a better predictor of lifespan than gene expression in multiple human tissues [64]. It remains to be seen, however, how will these markers compare to other markers such as the Horvath's epigenetic clock which can estimate chronological lifespan with 3–4 year accuracy [134]. It is possible though, that a combination of epigenetic and transcriptomic markers will form an even more accurate aging indicator.

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Functional Cell Biology

K. Umaer , ... N. Williams , in Encyclopedia of Cell Biology, 2016

Cleavage and Folding of Precursor rRNA

Ribosomal RNA processing has been studied extensively in the budding yeast, S. cerevisiae (reviewed in Woolford and Baserga, 2013; Nazar, 2004). In this organism, the major unit of the ribosomal RNA genes (rDNA) is organized in tandem consisting of the SSU gene (18S) at the 5′ end followed by two LSU genes (5.8S and 25S) at the 3′ end (Figure 2). There are approximately 150 repeats of this unit on chromosome XII. Not all copies are transcriptionally active at any given time, but the redundancy protects against genomic instability. The extra sequences at both ends of each tandem repeat that are transcribed but are not present in the final products are the external transcribed spacers, 5′ETS and 3′ETS. The sequences separating the rDNA genes which are also transcribed but excised during processing are the internal transcribed spacers, ITS1 (between 18S and 5.8S) and ITS2 (between 5.8S and 25S). Exonucleolytic and endonucleolytic activities are required to remove these sequences as indicated in Figure 2 (letters A to E, with subscripts added based on the overall chronology of events). Processing also involves chemical modification of bases at specific positions (as described below).

Figure 2. Processing of the major ribosomal RNA unit in S. cerevisiae. Sites of cleavage or exonucleolytic trimming are indicated with letters and subscript numbers. When known, enzymes responsible for these events are indicated in yellow ovals. The subcellular localization of different processing events is indicated on the right. There are two forms of 5.8S rRNA, 5.8Ss and 5.8SL which differ by 7 nucleotides at their 5′ ends. Subscript S and L denote small and long form of 5.8S rRNA. These two forms are generated by alternative pathways originating at 27SA2. 27SBs generates 5.8Ss and 27SBL generates 5.8SL. rRNA precursor species are named based on the sedimentation coefficient and the cut site that is used to generate the species.

Cleavage and modification of the precursor can start during transcription with about 80% of precursors being co-transcriptionally processed in yeast. The nascent RNA associates first with a series of ribonucleoprotein complexes collectively known as the SSU processome (Perez-Fernandez et al., 2007; Bernstein et al., 2004) in the nucleolus, required for the biogenesis of 18S rRNA. These RNPs have been classified according to their composition and time of assembly into several subcomplexes: the tUTP complex, the bUTP complex, the U3 snoRNP, the cUTP complex, the Bms/Rcl1 complex, and the Mpp10 complex. Later association with a different complex known as the A3 cluster is necessary for processing of the LSU rRNAs. The A3 cluster comprises the Pwp1 subcomplex, 6 interdependent A3 factors, and 2 Dead-box proteins. For a comprehensive list of yeast protein factors involved in ribosome assembly, including processing, see (Woolford and Baserga, 2013).

For a majority of transcripts, once the polymerase has transcribed ITS1, the first processing events occur on sites denoted A0 (on the distal end of the 5′ETS), A1 (on the proximal end of the 5′ETS), and A2 (in ITS1) (Figure 2). The last cleavage at site A2 separates the 18S precursor (20S) from the remaining nascent transcript. This second transcript, containing a portion of ITS1, 5.8S, ITS2, 25S, and 3′ETS, is released by cotranscriptional cleavage by the RNase III RntIp at the B0 site on the 3′ETS. Processing of the 3′ end of this precursor to site B2 generates 27SA2. Those transcripts that are not co-transcriptionally processed at A2 constitute the 35S pool. This 35S precursor is processed at the A0, A1, A2, and B2 sites, also generating 20S and 27SA2.

The enzyme responsible for the cleavages at A0, A1, and A2 is Rcl1p. This protein does not have a discernible in vitro activity on RNA, although it shares homology with 3′ terminal phosphate cyclase enzymes. Rcl1p associates with Bms1pn, and disruption of this association leads to accumulation of precursor (Delprato et al., 2014). Disrupting an interaction between protein factors Krr1 and Faf1 in the 90S complex impairs cleavage at sites A0, A1, and A2 (Zheng et al., 2014). The C-terminal domain of ribosomal biogenesis factor Rrp5 (Lebaron et al., 2013) is necessary for cleavage at A0 and A2 and its N-terminal domain is necessary for cleavage at A3. Rrp5 and Rok1, a helicase, make direct contact with RNA helices within expansion segment 6 of the SSU (Martin et al., 2014). The majority of 27SA2 precursors are cleaved at site A3 by endonuclease MRP (Mitochondrial RNA Processing) generating 27SA3. Exonucleases Rat1, Rrp17, and Xrn1 act in the 5′ to 3′ direction to trim this precursor to site B1S, generating intermediate 27SBS. This process requires Rat1 cofactor Rai1, as well as several other interdependent proteins belonging to the A3 factors. An alternative fate for 27SA2 is cleavage by an endonuclease at site B1L, generating a longer intermediate 27SBL. Eventually these two pathways generate two different pools of 5.8S in yeast cells, differing by ~7 nucleotides in their 5′ ends (Henry et al., 1994). Both are incorporated in ribosomes and are competent for translation. Approximately 80% of the 5.8S rRNA precursor gets processed through the 5.8SS pathway. These alternative forms have also been described in most but not all eukaryotes. Their significance is not clear.

No further cleavage occurs in the nucleolus. Intermediate 20S is transported to the cytoplasm as part of the pre-40S complex and cleaved at site D by Nob1p, assisted by Pno1/Dim2, generating its mature 3′ end. An additional event in cytoplasmic maturation of 18S involves rearrangement and formation of the 'beak,' a protrusion of helix 33 (Figure 1). This step involves Hrr25 dependent phosphorylation and dissociation of ribosomal protein Rps3, which is close to helix 33. After formation of the beak, Rps3 is reincorporated to the 40S subunit (Schafer et al., 2006).

RNA folding participates in the regulation of these cleavage events by ensuring the sequential nature of certain steps. Prior to cleavage at A2, folding of ITS1 prevents formation of helix 44 (Figure 1) at the 3′ end of the 18S sequence. Cleavage site D lies within this helix (Lamanna and Karbstein, 2011). Once the A2 site has been cut, this constraint on helix 44 is released, and Nob1p can recognize site D and generate the mature 3′ end of 18S.

Maturation of 27SBL and 27SBS continues in the nucleoplasm. A cleavage event within ITS2 at site C2 separates the 5.8S precursor (7SL or 7SS) from the 25S precursor (25.5S). Two conformations of ITS2 are possible: a ring structure and a hairpin structure. The ring structure may form first and facilitate protein association. The hairpin structure contains a stem (III) that is essential for processing. This stem brings together distant sequences and allows for base pairing interactions between 5.8S rRNA and 25S rRNA (Cote et al., 2002).

Exonucleolytic activity by Rat1p and Rrp17p in the 5′ to 3′ direction generates the mature 5′ end of 25S. Several 3′ to 5′ exonucleases of the exosome like Rrp4p (De La Cruz et al., 1998) act on the 3′ end of 7SL and 7SS, reducing it to a point 30 bases downstream of site E. Exonuclease. Rrp6p processes the remaining 30 nucleotides, generating mature 5.8SS or 5.8SL in the cytoplasm (Figure 2; Mitchell et al., 1996).

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