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Small RNA Research

A Brief Review of Small RNA Biogenesis

 

A. Small Regulatory RNAs
Small regulatory RNAs are non-coding RNA molecules that play important roles in activation or inhibition of cellular processes. They are 20-31 nt in length and interact with Argonaute family proteins to form effector ribonucleoprotein complexes. Three major classes of small regulatory RNAs have been identified to date: microRNAs (miRNA), PIWI-interacting RNAs (piRNA) and short-interfering RNAs (siRNA) (Table 1). Different kinds of small regulatory RNAs interact with distinct Argonaute proteins. Based on amino acid sequence similarities, Argonaute family proteins can be divided into two categories: AGO, named after its founding member in Arabidopsis thaliana, and PIWI, named after the Drosophila protein PIWI (P-element induced wimpy testis)(Carmell et al., 2002). AGO proteins are ubiquitously expressed in all the tissues and complex with miRNAs or siRNAs typically 20–23 nt in length (Bartel, 2004; Farazi et al., 2008; Ghildiyal and Zamore, 2009; Kim et al., 2009; Liu et al., 2008), whereas PIWI proteins are specifically expressed in germline cells.

Table 1 | Small Regulatory RNAs and Argonaute Family Proteins (Suzuki et al., 2012) 

  AGO PIWI
Expression All tissues Germline and cancer
Homologs Human Mouse Drosophila   AGO1, AGO2, AGO3, AGO4 AGO1, AGO2, AGO3, AGO4 AGO1, AGO2   HIWI, HILI, PIWIL3, HIWI2 MIWI, MILI, MIWI2 PIWI, AUB, AGO3
Bound small RNA miRNA siRNA piRNA
Length (nt) Precursor Biogenesis 3’ End 20–23 Hairpin-structured RNA Drosha, Dicer OH 20–23 dsRNA Dicer 2’-O-methyl 25–31 ssRNA Dicer-independent 2’-O-methyl
Mechanism of action ◆ Translational Repression
◆ mRNA degradation
◆ RNA cleavage ◆ Translational or post-transcriptional repression of transposons
◆ Multigenerational epigenetic phenomena in worms
Function Regulation of protein-coding genes Regulation of transposon, protein -coding genes, antiviral defense Regulation of transposon, unknown function

 

miRNAs and their biogenesis
miRNAs regulate post-transcriptional gene expression by RNA silencing in a wide range of eukaryotic organisms and viruses (Ambros, 2004; Bartel, 2004). While majority of miRNAs are located within the cell, some miRNAs, commonly known as circulating miRNA or extracellular miRNA, have also been found in extracellular environment, including various biological fluids and cell culture media.

miRNAs can origin from the introns or exons of protein-coding genes (about 30%) or the intergenic regions (70%) (Lin and Gregory, 2015). miRNAs are mainly transcribed by RNA polymerase II (Pol II) in the nucleus, and the primary miRNAs (pri-miRNAs) are capped, polyadenylated and spliced. The pri-miRNAs are several kilobases long and are processed in the nucleus by microprocessor, which includes DROSHA and DGCR8, to produce the 60-70 nucleotide precursor miRNAs (pre-miRNAs). The pre-miRNAs are then exported from the nucleus to the cytoplasm by XPO5 and its partner Ran-GTP. In cytoplasm, the pre-miRNAs are recognized by RNase III and DICER1 and further generate the mature miRNA duplexes. One strand of the mature miRNAs binds to miRNA-induced silencing complex (miRISC), which contains DICER1, AGO and miRNAs. The miRISC targets mRNAs by sequence base pairing and mediates mRNA degradation (Figure 1) (Lin and Gregory, 2015).

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Figure 1 | Schematic representation of miRNA biogenesis (Lin and Gregory, 2015).

As described above, DROSHA and DICER1 play vital roles in miRNA biogenesis pathway. However, they have lower-expression levels in some cancers, such as lung cancer, ovarian cancer and neuroblastoma. As a result, miRNA expression is globally suppressed in cancer cells compared with normal tissues (Lin and Gregory, 2015). Furthermore, DROSHA and DICER1 expression levels change along with the stage of tumor and can be used as a potential biomarker for neuroblastoma prognosis (Lin et al., 2010). Apart from microprocessor, other proteins involved in miRNA biogenesis are dysregulated in  kinds of certain cancers (Table 2). In addition, mutational analysis revealed that DROSHA is frequently mutated in Wilms tumours samples and ovarian cancers without affecting its expression levels. The alternatively spliced DROSHA transcripts are also found in melanoma and teratocarcinoma cells (Lin and Gregory, 2015). AGO proteins and their associated miRNAs are downregulated in activated CD4 T cells (Bronevetsky et al., 2013). The lack of DICER1 in the proximal epididymis causes dedifferentiation of the epithelium, leading to unbalanced sex steroid receptor expression, defects in epithelial lipid homeostasis, and subsequent male infertility (Bjorkgren and Sipila, 2015). Taken together, dysregulation of miRNAs biogenesis pathway not only alters miRNA production, but also causes diseases.

Table 2 | Dysregulation of miRNA biogenesis in cancers (Lin and Gregory, 2015).

Protein Dysregulation Cancer type Clinical correlation
DROSHA Upregulation Cervical SCC Altered miRNA profile; associated with neoplastic progression
Oesophageal cancer Regulates cell proliferation; associated with poor patient survival
BCC Not determined
SCC Not determined
Triple-negative breast cancer No clinical correlation
Smooth muscle tumours Associated with tumour progression
Gastric cancer Associated with pathological characteristics and patient survival
Serous ovarian carcinoma Associated with advanced tumour stages
Non-small cell lung cancer Associated with poor prognosis
Downregulation Bladder cancer Altered miRNA profile
Ovarian cancer Associated with poor patient survival
Endometrial cancer Correlated with histological grade
Nasopharyngeal carcinoma Correlated with shorter patient survival
Breast cancer Not determined
Gallbladder adenocarcinoma Correlated with metastasis, invasion and poor prognosis
Neuroblastoma Correlated with global downregulation of miRNAs and poor outcome
Cutaneous melanoma Associated with cancer progression and poor survival
DGCR8 Upregulation Oesophageal cancer Associated with poor patient survival
Bladder cancer Altered miRNA profile
SCC and BCC Not determined
Prostate cancer Associated with dysregulated miRNA
Colorectal carcinoma Not associated with any clinical parameters
Ovarian cancer Required for cell proliferation, migration and invasion
DICER1 Upregulation Smooth muscle tumours Associated with high-grade disease and tumour progression
Gastric cancer Correlated with gastric tumour subtype
Serous ovarian carcinoma Associated with advanced tumour stages
Prostate cancer Dysregulated miRNA expression; correlated with tumour stage
Oral cancer Required for proliferation
Colorectal cancer Correlated with tumour stage and associated with poor survival
Precursor lesions of lung adenocarcinoma Associated with histological subtypes and stages
Cutaneous melanoma Correlated with clinical stage
Downregulation Triple-negative breast cancer No clinical correlation
Bladder cancer Altered miRNA profile
BCC Not determined
Ovarian cancer Associated with advanced tumour stage and poor patient survival
Endometrial cancer No association with histological grade detected
Nasopharyngeal carcinoma Correlated with shorter patient survival
Breast cancer Associated with cancer progression and recurrence
Neuroblastoma Associated with global downregulation of miRNAs and poor outcome
Gallbladder adenocarcinoma Correlated with metastasis, invasion and poor prognosis
Non-small cell lung cancer Low levels of DICER1 expression correlate with shortened survival
Hepatocellular carcinoma Not associated with clinical characteristics
Chronic lymphocytic leukemia Associated with progression and prognosis
Colorectal cancer Associated with tumour stage and shorter survival
PACT Upregulation AK, SCC and BCC Not determined
XPO5 Downregulation Bladder cancer Associated with altered miRNA profile
AGO1 Upregulation AK, SCC and BCC Not determined
Serous ovarian carcinoma Associated with advanced tumour stages
AGO2 Upregulation AK, SCC and BCC Not determined
Serous ovarian carcinoma Correlated with advanced tumour stages and associated with shorter survival

Figure 2 | Biogenesis of piRNAs: the primary processing pathway in Drosophila (left) and the ping-pong pathway in mice (right).  

In Drosophila, nascent transcripts transcribed from piRNA clusters are processed into piRNA intermediates, which are then loaded onto PIWI proteins by Zucchini (Figure 2, left). During this step, the size of the bound PIWI proteins determines the length of mature piRNA. However, the factors involved in the transcription of piRNA clusters and its regulation remain elusive. Associated with PIWIs, the 3’ ends of piRNA intermediates are trimmed and subsequently 2’-O-methylated by Hen1/Pimet.

In mice, primary piRNAs are subjected to ping-pong pathway to enforce high levels of piRNA production in the germline cells (Figure 2, right). In the pathway, MILI associates with the primary piRNA, cleaves it with its endonuclease activity, and form the 5’ ends of secondary piRNAs. The cleavage products are then transferred onto MIWI2 and trimmed from the 3’ end to give rise to mature piRNAs. MIWI2 associated with the secondary piRNAs is localized to the nucleus upon piRNA loading. Therefore, MIWI2 does not contribute to the synthesis of secondary piRNAs via the ping-pong pathway. Like MIWI2, MIWI associated with pachytene piRNAs, which are expressed starting at the pachytene stage of meiosis in mouse spermatogenesis, is also barely involved in the ping-pong pathway. Instead, Aub and AGO3 play crucial roles in the secondary piRNAs synthesis. Like the primary piRNAs, secondary piRNAs are 2’-O-methylated by Hen1/Pimet (Ishizu et al., 2012; Iwasaki et al., 2015). 

Functionally, PIWIs associate with piRNAs to regulate transposon activity by RNA silencing or epigenetic regulation. It is reported that MILI and MIWI2 are needed for the methylation of genomic regions encoding transposons (Aravin et al., 2007; Kawaoka et al., 2008). PIWI physically binds heterochromatin protein 1 (HP1a) both in vivo and in vitro, leading to loss of heterochromatin. One common feature of defects in PIWI proteins is the increased DNA damage as measured by the foci of γ-H2Av, a histone H2A variant present at sites of dsDNA damage (Klattenhoff et al., 2007). MIWI and piRNAs are associated with polysomes, translation initiation factor eIF4E, and microRNA processor DICER, indicating multiple roles in regulation of protein translation and mRNAs stability (Grivna et al., 2006; Thomson and Lin, 2009).

PIWI proteins play crucial roles in meiosis. PIWIs mutations cause meiosis disorders. For example, MIWI2 mutants show predominant meiosis arrest at the leptotene stage (Thomson and Lin, 2009).

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Figure 3 | The expression time course of PIWI proteins during mammalian spermatogenesis. (Thomson and Lin, 2009).


PIWI protein expression is mostly restricted to the germline cells in different stages of the germline cycle (Figure 3). All three murine PIWI proteins, MIWI, MILI, and MIWI2, are expressed during spermatogenesis. More specifically, MIWI2 is expressed from 15.5 dpc to 3 dpp in mitotically arrested prenatal germline stem cells (GSC), although also in Sertoli cells not necessary for a germline function. MILI expression is detected from 3 dpp in mitotically arrested prenatal GSCs to round spermatids. MIWI is merely expressed from meiotic spermatocytes to elongating spermatids. 

Furthermore, PIWIs is not only expressed in germline, but also detected in somatic tissues. In Drosophila somatic tissues such as salivary glands and eyes, PIWIs bind to chromosomes and are associated with epigenetic effects at the binding sites. In planaria, PIWI proteins are expressed in neoblasts that are capable of tissue regeneration. In human, HIWI is detected in hematopoietic stem cells but not in the downstream progenitors and mature immune cells. Particularly, PIWIs are expressed in a wide variety of human cancers (Table 3)(Suzuki et al., 2012). For example, the expression of HIWI is positively correlated with glioma grade: high HIWI expression has poorer clinical outcomes (Sun et al., 2011), although it is unclear whether or how the piRNAs are involved in this process.

Table 3 | PIWI expression in human cancers (Suzuki et al., 2012)

Disease Material PIWI Method
Breast cancer Tissue, MDA-MB-231 HILI RT-PCR, RNA array, WB, IC
Breast cancer Tissue HILI IHC
Breast, cervical, and other cancers MDA-MB-231, MDA-MB-468, MCF-7, HeLa, THP-1, CCRF, Jurkat, H9, Raji, Daudi, HEL, Dami, HL-60, K562, PBL985, HCT-8, 3B11, CaoV3, CaCo, HT-29, SW480, Huh7, CT26CL25, Hey1B, SW872, H1299, C8161, CT26CL25, Hey1B, SW872, H1299, C8161, HepG2, INS-1, LL2, N2a HILI, PL2L50, PL2L60, PL2L80 RT-PCR, WB, IHC
Cervical cancer Tissue HIWI IHC
Cervical cancer Tissue HILI IHC
Cervical cancer HeLa HILI WB
Colon cancer Tissue HIWI IHC
Colorectal and other cancers Human tissue, 823, AGS, N87, GES1, E30, E70, E140, E180, E410, HepG2, 7402, 7721, YES2, T12, LoVo, CL187, HT-29, RKO, SW480, HCT116, PG, GLC82, H446, H460, H1299, A549 HIWI WB, IHC
Endometrial cancer Tissue HIWI IHC
Esophageal cancer Tissue, KYSE70, KYSE140, KYSE450 HIWI WB, IC, IHC
Gastric cancer Tissue HIWI, HILI, PIWIL3, HIWI2 IHC
Gastric cancer Tissue, AGS, NCI-N87, SNU-1, SNU-5, SNU-16 HIWI RT-PCR, IHC, WB
Glioma Tissue, U251, U87, LN229 HIWI RT-PCR, WB, IHC
Liver cancer Tissue, HepG2, SMMC7721, MHCC97L, MHCC97H, HCCLM3 HIWI qRT-PCR, WB, IHC
Ovarian cancer A2780, CP70, CDDP, MCP2, MCP3, MCP8, 2008, 2008C13 HILI WB
Pancreatic cancer Tissue HIWI qRT-PCR, IHC
Sarcoma Tissue HIWI qRT-PCR
Sarcoma Tissue, MFH HIWI IHC
Seminoma Tissue HIWI qRT-PCR
Seminoma and other cancers Tissue, MDA-MB-231 HILI RT-PCR, IC, IHC

 

siRNAs and their biogenesis
Short interfering RNAs (siRNAs), also known as small interfering RNAs or silencing RNAs, are a class of double-stranded RNA molecules, 20-23 nt in length. Like miRNAs, siRNAs cleave and degrade target mRNAs guided by base pairing in the RNA interference (RNAi) pathway (Agrawal et al., 2003). Moreover, some siRNAs can base pair with DNA and induce DNA methylation to regulate gene expression (Kawasaki and Taira, 2004). Furthermore, siRNAs also play crucial roles in virus defense and chromatin remodeling.

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Figure 4 | Biogenesis of siRNAs in mammals. 

Exogenous siRNAs (exo-siRNAs) are originated from foreign dsRNAs taken up from outside the cells, for example, virus infection and therapeutic dsRNAs. Endogenous siRNAs (endo-siRNAs), on the other hand, arise from genomic loci, such as centromeres, transposons, and inverted repetitive sequences that produce transcripts capable of forming dsRNA structures. Bidirectional transcription, antisense transcripts, or pseudogenes at the same (cis-nat-siRNA clusters) or different loci (trans-nat-siRNA clusters) are also the sources of the dsRNAs (Carthew and Sontheimer, 2009; Watanabe et al., 2008). Although both endo-siRNAs and miRNAs function as RISC, miRNAs arise from the ~60-70 nt precursors of intramolecular stem-loop structures that lack perfect Watson-Crick base pairing, whereas siRNAs can be processed from duplex structures that are perfectly base paired (Golden et al., 2008). The other significant difference is that siRNAs carry 2’-O-methyl modifications at their 3’ termini, whereas miRNAs do not (Table 1). 

Exo-siRNAs derived from exogenous dsRNAs are processed by Dicer and Dicer binding proteins (Dicer-TRBP or Dicer–PACT). The processing of endo-siRNAs also requires Dicer, however the role of TRBP and PACT remains undetermined. Mature endo-siRNAs are loaded onto Argonaute 2 (AGO2). Whether endo-siRNAs are loaded onto other AGO members, such as AGO1, AGO3 and AGO4, remains to be determined. In mammals, exo-siRNAs are loaded onto AGO1, AGO2, AGO3 and AGO4; however, only the AGO2–siRNA complex functions in RNA interference, as other AGO members lack Slicer activity (Siomi et al., 2011)(Figure 4). 

The main function of siRNAs is gene silencing via mRNA cleavage by RNA-induced Silencing Complex (RISC). siRNAs involved in DNA methylattion also play a crucial role in chromatin remodeling. Biologically, endo-siRNAs keep tabs on domestic miscreants such as transposons, whereas exogenous siRNAs are called upon to defend against foreign opponents such as viruses (Golden et al., 2008). 

In Drosophila, mutants of key protein components in the siRNA silencing pathway are viable and fertile, but highly susceptible to virus infection (Marques and Carthew, 2007). Additionally, transposon mRNAs are increased 2 ~ 10-fold in the heads and ovaries of AGO2 mutants, in the heads of DICER2, and in the S2 cells with DICER2 or AGO2 knockdown. However, no significant increase in transposon transcripts was apparent after DICER1 knockdown (Marques and Carthew, 2007). The levels of the 3’ overlapping transcripts Pdzd11 and Kif4 in the cis-nat-siRNA cluster increased modestly in DICER mutant mice (Watanabe et al., 2008). 


B. Other Types of Small RNAs
Apart from small regulatory RNAs, there are diverse small RNAs playing vital roles in cellular activities and human diseases. Transfer RNA (tRNA), small nucleolar RNA (snoRNA) and small nuclear RNA (snRNA) have well established canonical functions and have received increasing attention for their new non-canonical biological activities recently. As a new class of small RNAs, tRNA-related fragments (tRF) and tRNA halves (tiRNA) are generated from tRNAs via precise biogenesis mechanism, having functions different from the parent tRNAs.

tRNAs 
The principal function of tRNAs is decoding mRNAs and protein translation. tRNAs are composed of 73~95 nucleotides and made up of the D-loop, T-loop, anticodon loop and variable loop in the cloverleaf structure representation. Alteration of tRNA repertoire affects mRNA translation efficiency (Gorochowski et al., 2015) and mRNA stability (Gingold et al., 2014). tRNA-related fragments (tRF) and tRNA halves (tiRNA) are derived from tRNAs by angiogenin (ANG), Dicer, and other nuclease cleavages. This new class of small RNAs has recently gained discovered for their surprising roles in biological processes and human diseases (Fu et al., 2015; Kirchner and Ignatova, 2015; Raina and Ibba, 2014).

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Figure 5 | Biogenesis and functions of tRNA, tRF and tiRNA.

tRNA biogenesis comprises multiple processes of transcription, processing, splicing, chemical group modification, CCA addition, nuclear-to-cytoplasmic transportation, and aminoacylation. tRNA precursors (pre-tRNA) are transcribed from individual tRNA genes by RNA polymerase III (Pol III), transcription factor TFIIIC and TFIIIB (consist of BDP1, BRF1 and TBP) (Figure 5). The pre-tRNAs are removed of the 50- and 30-nt trailers by endonucleases RNase P and RNase Z, modified with chemical groups, and added with the terminal CCA by nucleotidyl transferase. Finally, the mature tRNAs are exported to the cytoplasm by exportin-T (XPOT) to participate in protein synthesis (Phizicky and Hopper, 2010). Accurate amino acid charging by aminoacylation is catalyzed by aminoacyl tRNA synthetases (aaRS).

The diverse and extensive post-transcriptional alterations of pre-tRNAs are one of the most striking features of tRNA biogenesis and maturation. RNA editing and alternative splicing are highest in the brain compared with other tissues, which has important implications in the central nervous system (CNS). Polyribonucleotide 5'-hydroxyl-kinase CLP1 is an enzyme in the steps of tRNA intron splicing. CLP1 mutation causes accumulation of toxic pre-tRNA splice intermediate, leading to motor sensory neuropathy and neurological diseases (Hanada et al., 2013; Karaca et al., 2014; Schaffer et al., 2014). Additionally, GCN2, a kinase played special role in tRNA metabolism, has an essential role in supporting tumor cell growth and proliferation (Anderson and Ivanov, 2014).
tRNA modifications are involved in all aspects of tRNA biochemistry, from secondary and tertiary structures, precise recognition by aminoacyl-tRNA synthetases, to mRNA decoding. Aberrant tRNA modifiers and tRNA modifications are linked to human diseases such as cancer, Type 2 diabetes, neurological disorders, and mitochondrial disorders (Torres et al., 2014). For example, tRNA methyltransferase 12 homolog (TRMT12), one of the enzymes that catalyze wybutosine modification at position 37 on tRNAPhe, is amplified and over expressed in breast cancer cell lines tumors.

tRNA derived fragments (tRF) and tRNA halved (tiRNA)
tRFs are derived from tRNAs by precise enzymatic cleavages by endonucleases angiogenin (ANG) and Dicer to produce tRF-5 and tRF-3 (Figure 5). Further, tRNA-splicing endonuclease (TSEN) complex excises the pre-tRNAs and produces i-tRF in nucleus (Anderson and Ivanov, 2014) (Figure 5). Additionally, tRF-1 is generated from the 3’ tails of pre-tRNAs by Dicer, RNase Z, and zinc phosphodiesterase ELAC protein 2 (ELAC2). tiRNAs are tRNA halves generated by angiogenin cleavage in the anticodon loop of mature tRNAs.

tRFs and tiRNAs are not random degradation products of tRNAs. Rather they are a class of bioactive functional small RNAs. They are known to act as microRNAs in RNA interference; directly inhibit protein synthesis by displacing eIF4G translation initiation factor eIF4G from mRNA on ribosomes [9-10]; bind protein factors such as CBX1 to regulate target mRNA stability; interact with cytochrome c to modulate apoptosis; assemble stress granules in response to stress conditions; sensitize cells to oxidative-stress-induced p53 activation and p53-dependent cell death; alter transcriptional cascades in intergenerational inheritance as paternal epigenetic factors. Clinically, tRF/tiRNAs are associated with or are causal factors for disease conditions including cancers, neurodegeneration, and metabolic disorders. Due to the high enrichment in biofluids, sometimes more so than microRNAs, tRF/tiRNA populations have many desired properties as biomarkers. For example, the tRF profiles have been shown to discriminate triple-negative, triple positive breast cancer cells from the normal controls.

snRNAs/snoRNAs and their biogenesis
Small nuclear RNAs (snRNA) and small nucleolar RNAs (snoRNAs) are two well-studied classes of ncRNAs in the form of RNPs. snRNPs form the core of the spliceosome and catalyze the removal of introns from pre-mRNA. snoRNAs are involved in the modification and processing of pre-ribosomal RNAs. Additionally, snoRNAs are essential for major biological processes including protein translation, mRNA splicing and genome stability (Dragon et al., 2006; Matera et al., 2007).

According to the common sequence features and protein cofactors, the snRNAs can be divided into two classes: Sm- and Lsm-class snRNAs (Figure 6A,B) (Matera et al., 2007). Sm-class genes are transcribed by a specialized form of RNA polymerase II (Pol II). Integrator complex subunits INT9 and INT11 are essential for the proper cleavage and polyadenylation of the 3’ ends of snRNAs. Following transcription and 3’ processing in the nucleus, Sm-class snRNAs are transported to the cytoplasm by an export complex that contains phosphorylated adaptor for RNA export (PHAX), exportin 1 (CRM1), cap binding complex protein (CBC), and Ran GTPase (Figure 7). The export complex dissociates from the pre-snRNA in the cytoplasm after binding with the assemblyosome SMN complex. The SMN complex recruits a set of seven Sm proteins to form-core RNP. Following assembly of the Sm core, the m7G cap is hypermethylated by TGS1 to form a 2,2,7-trimethylguanosine (TMG) cap structure, and the 3’ end is trimmed by an unknown exonuclease. Triggered by TMG cap, SPN associates with Imp-β to assemble the nuclear import complex. After transported into the nucleus, the Sm-class snRNPs target to Cajal bodies for snRNP maturation. Additional RNP remodeling and assembly steps are thought to take place in Cajal bodies, including RNA-guided modification of the spliceosomal snRNAs and assembly of factors that are specific to a given species of snRNP. Finally, the newly minted snRNPs either participate in splicing at perichromatin fibrils (PFs) or are stored in interchromatin granule clusters (IGCs) for later use. With great difficulty, Lsm-class snRNA genes are transcribed by Pol III using specialized external promoters. The run of uridines that forms the Lsm binding site at the 3′ end also doubles as a Pol III transcription terminator. Therefore, there are few parallels between Lsm-class genes and protein-coding genes. Additionally, Lsm-class snRNAs never leave the nucleus during the maturation process(Matera et al., 2007).

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Figure 6 | The features of snRNAs and snoRNAs (Matera et al., 2007). (A) Sm-class RNAs typically contain a 5’-trimethylguanosine cap, a 3’ stem-loop and a Sm proteins binding site. (B) Lsm-class RNAs are characterized a monomethylphosphate cap and a 3’ stem-loop, terminating in a Lsm proteins binding site that is made up of a stretch of uridines. (C) C/D RNAs are characterized by conserved motifs C and D that form a kink-turn (K-turn) or alternate boxes C’ and D’ (orange) that could also form a K-turn; 2’-O-ribose methylation is performed on the rRNA residue that is base-paired to the fifth position upstream from box D (or D’). (D) H/ACA RNAs adopt a hairpin-hinge-hairpin-tail structure where box H is found in the hinge region and box ACA is found three nucleotides upstream of the 3’- end; each hairpin usually contains an internal loop called pseudouridylation pocket where C formation in rRNA occurs on the first unpaired U residue upstream from box H or ACA.

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Figure 7 | Biogenesis of Sm-class snRNPs (Matera et al., 2007)
Similarly, snoRNAs can be grouped into two major families on the basis of conserved sequence motifs: C/D and H/ACA snoRNAs (Figure 6C,D) (Dragon et al., 2006). C/D RNAs direct 2’-O-ribose methylation, whereas H/ACA RNAs guide pseudouridylation. Additionally, other modification targets include snRNAs in eukaryotes, tRNAs in archaea, spliced leader RNAs in trypanosomes and perhaps at least one brain-specific mRNA in mammals. Spliceosome function also depends on the modification of snRNAs by C/D and H/ACA RNAs. Moreover, a H/ACA telomerase RNA is required for telomere synthesis.

snoRNAs are predominantly located in introns of mRNAs and transcribed by pol II (Figure 8). During the transcription, three of the four core H/ACA RNP proteins, DKC1, Nop10 and Nhp2, and an assembly factor Naf1 associate with the snoRNAs. However, the core RNP proteins of C/D RNP include fibrillarin, NHPX, NOP56 and NOP58, which are different from H/ACA RNPs’. Naf1 ensures the assembly of a stable H/ACA pre-RNP which is inactive until Naf1 is exchanged for Gar1. Naf1 is required only for the accumulation of all classes of H/ACA RNA, whereas Gar1 is essential for the function of H/ACA RNPs. The assembly of snoRNPs seems to occur co-transcriptionally and to be tightly coupled to pre-mRNA splicing by IBP160 (Figure 8). For C/D RNAs, the assembly of C/D RNPs requires a potential exchange factor Bcd1 that acts the same role as Naf1 in H/ACA RNPs assembly. Both C/D and H/ACA RNPs are rapidly targeted to Cajal bodies where their essential maturation steps occur. Additionally, PHAX may help to localize certain C/D and H/ACA RNAs to Cajal bodies for RNA modification and RNP assembly, where TGS1 and SMN complex are two important components. Depending on their functions, the C/D and H/ACA RNAs ultimately localize to nucleoli, Cajal bodies or telomeres (Esteller, 2011; Matera et al., 2007). 

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Figure 8 | Biogenesis of snoRNPs.

As described above, the C/D RNPs consist of a core of four proteins — fibrillarin, NOP56, NOP58 and NHP2L1 — whereas the H/ACA RNPs contain DCK1, GAR1, NHP2 and NOP10. Fibrillarin is essential for development, and its depletion is lethal in embryos. Mutations in the human DKC1, NOP10 and NHP2 genes are associated with the X-linked genetic disorder dyskeratosis congenita, which is susceptible to epithelial cancers. Biallelic null mutations in U6 snRNA biogenesis phosphodiesterase 1 (Usb1) cause poikiloderma with neutropenia (PN), which is predisposed to developing myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). In S. pombe, loss of Usb1 (Mpn1) function leads to an increase in TERRA telomere transcripts and decrease in telomere length. Disorder of Cajal bodies, a subnuclear structure for snRNA and snoRNA maturation, occurs in human pathologies including cancers, inherited neurodegenerative diseases, aberrant cellular proliferation, cell cycle, stress response, and aging (Cioce and Lamond, 2005; Mroczek and Dziembowski, 2013; Stepanov et al., 2015).

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