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Small nuclear RNA (snRNA) –Types, Structure, Steps, Functions

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Small nuclear RNA (snRNA) is one of the small RNA with an average size of 150 nt. Eukaryotic genomes code for a variety of non-coding RNAs and snRNA is a class of highly abundant RNA, localized in the nucleus with important functions in intron splicing and other RNA processing. Generally, in transcript splicing, snRNA presents as a ribonucleoprotein particles (snRNPs) along with additional proteins that form a large particulate complex (spliceosome) bound to the unspliced primary RNA transcripts in order to mediate the process. Besides splicing, additional evidence indicate snRNPs function in nuclear maturation of primary transcripts in mRNAs, gene expression regulation, splice donor in non-canonical systems and in 3′-end processing of replication-dependent histone mRNAs.

Small nuclear RNAs (snRNAs) are critical components of the spliceosome that catalyze the splicing of pre-mRNA. snRNAs are each complexed with many proteins to form RNA-protein complexes, termed as small nuclear ribonucleoproteins (snRNPs), in the cell nucleus. snRNPs participate in pre-mRNA splicing by recognizing the critical sequence elements present in the introns, thereby forming active spliceosomes. The recognition is achieved primarily by base-pairing interactions (or nucleotide-nucleotide contact) between snRNAs and pre-mRNA. Notably, snRNAs are extensively modified with different RNA modifications, which confer unique properties to the RNAs.


What is Small nuclear RNA (snRNA)?

  • A variety of noncoding RNAs are encoded by eukaryotic genomes, and snRNA is a kind of highly abundant, nucleus-localized RNA with essential roles in intron splicing and other RNA processing.
  • In order to mediate the process of transcript splicing, snRNA typically exists as ribonucleoprotein particles (snRNPs) with other proteins that create a huge particulate complex (spliceosome) bound to the unspliced primary RNA transcripts.
  • In addition to splicing, snRNPs function in nuclear maturation of initial transcripts in mRNAs, gene expression regulation, splice donor in non-canonical systems, and 3′-end processing of replication-dependent histone mRNAs, according to new data.
  • Intronless, non-polyadenylated, non-coding transcripts with nuclear function.
  • Small nuclear ribonucleoprotein complexes are always composed of SnRNA and a set of particular proteins (snRNP often pronounced “snurps”)
  • Each snRNP particle consists of snRNA and multiple snRNP-specific proteins.


Types of Small nuclear RNA (snRNA)

The snRNAs can be divided into two classes on the basis of common sequence  features and protein cofactors.

1. Sm-class RNAS

  • This is characterised by a 5′-trimethylguanosine cap, a 3′ stem-loop, and a binding site for a heteroheptameric ring-shaped group of seven Sm proteins (the Sm site).
  • It includes U1, U2, U4,U4atc, U5, U7, U11, and U12 teams.
  • The RNA polymerase II (Pol II) used to transcribe Sm-class genes is functionally comparable to the Pol II used to transcribe mammalian protein-coding genes.
  • Sm-class snRNAs are exported from the nucleus to undergo maturation in the cytoplasm.

2. Lsm-class RNAs

  • This class possesses a monomethyl phosphate cap and a 3′ stem-loop, the latter of which terminates in a stretch of uridines that serve as the binding site for an unique heteroheptameric ring of Lsm proteins.
  • It contains U6 and Ubatac
  • The Lsm-class snRNA genes (U6 and Ubatac) are transcribed by Pol III using external Promoters that are specialised for this purpose.
  • snRNAs of the Lsm class never leave the nucleus.


Structure of snRNA

  • Distal sequence element (DSE) works as an enhancer.
  • Required connection between the 3′ box RNA processing element and the promoter of the snRNA gene type.
  • In addition to a DSE and PSE, the pol III-transcribed genes have a TATA box at the -25 location.
  • The core promoter is an important snRNA gene-specific proximal sequence element (PSE).


Steps of snRNA gene transcription

phosphorylation of the CTD of Pol II during snRNA gene transcription. Initial phosphorylation of Ser5 and Ser7 by the cyclin-dependent kinase (CDK)7 subunit of TFIIH. Ser7P interacts with RPAP2. The Integrator subunits Int1, Int4, Int5, Int6, and Int7 are recruited by RPAP2. Unknown mechanism recruits positive-transcription elongation factor b (P-TEFb) after RPAP2 dephosphorylates Ser5P. CDK9, a subunit of P-TEFb, phosphorylates Ser2. The double phosphorylation of Ser2 and Ser7 recruits the RNA processing enzyme Int9/11.


Transcription termination of the snRNA gene

Model for the termination of U2 snRNA gene transcription. The snRNA transcript is shown in green with a cap at the 5′ end, whereas nucleosomes are depicted as barrels. Pol Il continues transcription after recognising the 3′ box, whereas Integrator processes the newly synthesised RNA. CTCF identifies the CTCF binding site upstream of the 3′ box and regulates nucleosome occupancy. Negative elongation factor (NELF) is recruited by DRB sensitivity-inducing factor (DSIF) and CTCF at the end of the transcription unit and causes transcription termination


Functions of modifications in snRNAs

On a molecular level, RNA modifications alter the properties of the four basic nucleotides, thereby influencing the inter- and intra-molecular interactions of the RNAs that carry them. 2′-O-methylation typically stabilises RNA helices by increasing base-stacking whereas pseudouridine has a higher hydrogen bonding capacity than uridine and also increases the rigidity of the sugar-phosphate backbone. These chemical and topological properties explain the prevalence of these particular modifications in the highly structured snRNAs that are required to form numerous critical RNA-RNA interactions. In contrast, m6A can have diverse effects on RNA secondary structure by either destabilising RNA duplexes or, when present in single-stranded RNAs, promoting base stacking, thereby enhancing RNA stability. m2G has only a minimally stabilising effect on RNA structure but is proposed to alter the base-pairing interactions compared to guanosine.

It is possible that modifications in snRNAs arise due to the high concentration of modification enzymes in the nucleus and the accessibility of particular snRNA sequences. However, the presence of a dedicated snRNA modification machinery, together with the clustering of snRNA modifications in functionally important sequences that form key RNA-RNA or RNA-protein interactions, instead implies that snRNA modifications likely serve to fine-tune these interactions to optimise the efficiency and fidelity of pre-mRNA splicing. For example, in the context of the di- and tri-snRNPs, the U4 and U6 snRNAs are extensively basepaired, and approximately half of the modified nucleotides in these snRNAs are present in sequences involved in establishing these interactions. Dissociation of the U4-U6 basepairing by the RNA helicase Brr2 is a key event that is proposed to serve as a proof-reading step during formation of the catalytically active spliceosome and it is tempting to speculate that modifications within the basepaired sequences influence the kinetics of RNA unwinding and re-assembly. Likewise, within pre-catalytic and catalytic spliceosomes, the U6 snRNA forms extensive and dynamic interactions with the U2 snRNA, and a myriad of modified nucleotides lie within the sequences involved in basepairing. In pre-catalytic spliceosomes, U6 and U2 are proposed to form three- and a four-helix junctions and single-molecule Förster resonance energy transfer (smFRET) analyses indicate a dynamic equilibrium between different conformations in human cells. Post-transcriptional modifications (for example, Ψ15, Gm11, Gm12 and Gm19 in humans) within the human U2 stem I are suggested to regulate these conformational changes and to stabilise the four-helix structure. However, the effects observed in this in vitro reconstituted system are relatively mild, leading to the suggestion that these modifications may not only modulate snRNA-snRNA interactions but may also influence snRNA-protein binding in vivo.

Alongside potentially modulating snRNA-snRNA interactions, RNA modifications may also influence basepairing interactions between snRNAs and their pre-mRNA substrates. The high density of modifications within such sequences is exemplified by the 5′ region of the U1 snRNA that contains two cap-proximal 2′-O-methylations (U1-Am1 and U1-Um2 in humans) and two evolutionarily conserved Ψ (U1-Ψ5 and U1-Ψ6 in humans) and which basepairs with the 5′SS in the spliceosomal E and A complexes. Although 5′SS selection does not solely rely on complementarity to the 5′ end of the U1 snRNA, the strength of U1-5′SS basepairing is suggested to correlate to some extent with 5′SS usage. The presence of the two U1 Ψ’s leads to formation of A-Ψ and R-Ψ wobble basepairs and the effects of modifications/mutations within these sequences have been extensively studied. Early work indicated that ‘suppressor U1 snRNAs’, which restore perfect basepairing with strong 5′SSs, had little or no effect on 5′SS usage, questioning the importance of Ψs at these sites. Furthermore, in vitro thermodynamic analyses demonstrated that the substitution of pseudouridine for uridine does not influence the free energy of duplex formation between the sequence at the 5′ end of U1 and a complementary RNA strand. More recently, however, positive effects of ‘suppressor U1 snRNAs’ were reported and in vitro competition assays suggested that the presence of G-Ψ basepairs at the 5′ end of U1 can be advantageous for 5′SS selection. While the precise role of the U1 5′ end Ψs therefore remains unclear, it is possible that they have minimal influence on the interactions between the U1 snRNA and strong 5′SS but that they may effect basepairing of U1 with weaker 5′SS.

Similarly, a human U6 sequence containing three Nm, one Ψ (U6-Ψ40) and 1 m6A (U6-m6A43) binds the 5′SS during the subsequent catalytic phase of splicing. In yeast, mutations within the U6 sequence that binds the 5′SS are lethal, highlighting the critical nature of this sequence for pre-mRNA splicing and raising the possibility that modifications within this region could play important roles in regulating the formation or stability of U6-5′SS basepairing. Interestingly, the ACm6AGAGA sequence of U6 is contacted by U5 snRNP protein PRP8, which plays a crucial role in the formation of the catalytic core of the spliceosome, and it is possible that the presence of U6-m6A43 modulates this interaction. This model is supported by the finding that the expression levels of the m6A methyltransferase METTL16 and PRP8 are co-regulated in human cells. Interestingly, PRP8 also binds to an evolutionarily conserved 11 nucleotide loop in the U5 snRNA, which contains three Nm (U5-Gm37, U5-Um41 and U5-Cm45) and two Ψ (U5-Ψ43 and U5-Ψ46) in humans, and directly contacts the 5′ exon and 3′ exon of pre-mRNAs during the first and second catalytic steps of splicing respectively. While experimental evidence demonstrating the importance of these modifications for splicing is still lacking, it is possible that they either influence directly the U5 snRNA-pre-mRNA basepairing or that they are required for the PRP8-mediated stabilisation of exon-U5 loop1 interactions during splicing.

U2 sequences that basepairs with the pre-mRNA BSS are also highly modified and modified nucleotides within such sequences have been suggested to be important for U2 snRNP biogenesis, spliceosome assembly, pre-mRNA interaction and also the catalytic activity of the spliceosome. Yeast U2 lacking Ψ within the branch site recognition region (BSRR) can assemble to form a non-functional 12S pre-U2 snRNP particle but cannot progress to the functional 17S complex and consequently, pre-mRNA splicing is abolished leading to growth defects. In humans, modifications at the 5′ end of U2 are required for formation of the spliceosomal E complex and while U2-Am1, U2-Um2, U2-Gm12 and U2-Gm19 are individually essential for pre-mRNA splicing, the Ψs within this region are only collectively required. It has further been shown in yeast that the RNA helicase Prp5 binds to U2 lacking Ψ42 and Ψ44 within the BSRR with significantly lower affinity than the modified form and that consequently, the RNA-dependent ATPase activity of Prp5 is reduced. This implies that the progression of spliceosome assembly from the early (E) complex to complex A is impeded in the absence of U2 pseudouridylation. When U2 is basepaired with the pre-mRNA, Ψ34 (human) or Ψ35 (yeast) directly opposes the branch point nucleotide, which mediates nucleophilic attack on the 5′SS during the first catalytic step. Excitingly, structural data from yeast have revealed that presence of the U2 Ψ plays an important role in precisely positioning the nucleophile to allow the catalytic reaction to take place.

There is a marked contrast between the number of modifications reported in the snRNAs of the major and minor spliceosomes, with no modifications so far detected in the human U11 snRNA, and only very few identified in the U4atac, U6atac and U12 snRNAs. As the snRNAs of the minor splicoeosome are present in the cell at much lower levels than those of the major spliceosome, it is possible that this apparent difference may arise from the technical challenges of detecting, potentially sub-stoichiometric, modifications in low abundance RNAs. However, the recent application of modification mapping approaches such as RiboMeth-seq and m6A-seq, which are coupled to next generation sequencing and therefore highly sensitive, have not provided new evidence for additional modifications in these RNA species, suggesting a bona fide difference in the extent of modification of the major and minor snRNAs. Compared to major U2-type introns, the 5′SS and BSS of U12-type introns removed by the minor spliceosome have significantly lower sequence diversity and consequently, it is possible that a lower degree of modification is required in the snRNAs of the minor spliceosome. This hypothesis is supported by the observation that in yeast, where there is less variation in the sequences of the pre-mRNA splice sites, the snRNAs are less modified than in humans.


The stoichiometry and dynamics of snRNA modifications

The development of quantitative techniques for the detection of RNA modifications have recently provided the first insights into the stoichiometry of modifications in abundant RNAs and together with the discovery of demethylases that can act as modification ‘erasers’, these studies have highlighted the dynamic nature of RNA modifications and emphasised their potential as important regulators of gene expression. A recent analysis of snRNA 2′-O-methylation demonstrated that all canonical sites are almost fully modified in diverse human tissues and it is anticipated that the vast majority of other known snRNA modifications are also constitutively present. However, alterations in the modification status of snRNAs have also been reported, further supporting the model that modifications at specific snRNA positions can have important physiological roles.

In yeast, two additional Ψ at positions 56 and 93 of the U2 snRNA are detected in nutrient-deprived cells, and Ψ56 is also observed upon exposure of cells to heat shock. These inducible modifications are installed by the action of the stand-alone pseudouridine synthetase Pus7 (U2-Ψ56) and the H/ACA box snoRNP snR81 that normally modifies U1051 in the 25S ribosomal RNA (U2-Ψ93). Interestingly, U2-U56 lies within a sequence context that is similar to the typical Pus7 consensus motif and the sequences proximal to U2-U93 have similar, but imperfect, complementarity to the snR81 guide sequences; it is therefore suggested that under stress conditions, the recognition criteria of Pus7 and snR81 become less stringent enabling these additional sites to be targeted. Using reporter systems, it has been shown that the presence of U2-Ψ93 impedes pre-mRNA splicing and mechanistically, it is suggested that Ψ56 and Ψ93, which lie within stem IIa and stem IIc of U2, may influence the kinetics of U2 switching between its optimal substrate-interaction conformation and its catalytic conformation, thereby negatively affecting pre-mRNA splicing. Recently, smFRET experiments demonstrate that, compared to U93-containing RNAs, the presence of Ψ93 increases the conformational flexibility of the stem IIc sequence, whereas Ψ56 reduces the conformational dynamics of stem II and stabilises stem IIc. A further example of inducible snRNA modification is that during filamentous growth of S. cerevisiae, U6-U28 is pseudouridylated by Pus1. Interestingly, it has been shown that installation of this additional modification is an important aspect of the filamentous growth programme. It is proposed that U6-Ψ28 may influence the recruitment or binding of Cwc2, which is a critical step during catalytic activation of the spliceosome, and that this may in turn affect the splicing of particular pre-mRNAs that encode proteins required for filament formation. Likewise, changes in the extent of 2′-O-methylation of specific snRNA nucleotides (cap+2 of U4 and U5 and various internal sites in U2 and U6) have also been observed during human T cell activation. Compared to primary T cells, a generally lower level of snRNA 2′-O-methylation was detected in Jurkat cells, a common T cell leukaemia model, and it is suggested that due to the very high growth and RNA synthesis rates in these cells, the modification machinery becomes limiting, leading to substoichoimetric modification of diverse RNA species. In contrast to most human tissues, the U4 snRNA in Jurkat cells lacks the Cm8 modification. In the free U4 snRNP, Cm8 likely contributes to stabilising a short intra-molecular helix whereas in the context of the U4/U6.U5 tri-snRNP, U4-Cm8 lies within a long stretch of basepairing with the U6 snRNA. It is possible, therefore, that the absence of U4-Cm8 promotes incorporation of U4 into the tri-RNP, thereby increasing the efficiency of spliceosome assembly and pre-mRNA splicing in Jurkat cells.

The presence of both constitutive and inducible modifications in snRNAs raises the intriguing possibility that the constitutive modifications represent a core subset that are involved in fundamental aspects of spliceosome assembly or function, whereas inducible modifications likely subtly alter the stability, kinetics or dynamics of interactions within spliceosomal complexes to promote or impede splicing of particular target pre-mRNAs. Interestingly, a recent report indicates that changes in snRNA modifications may not only arise at the level of modification installation. It is proposed that in addition to 2′-O-methylation by CMTR1, the cap+1 nucleotides of the RNA pol II synthesised snRNAs also undergo N6-methylation, leading to the formation of m6Am, but that in some cell lines and tissues, these base modifications are efficiently removed by the demethylase FTO. Cap proximal m6Am is suggested to promote snRNA stability and increased exon inclusion is observed in cells lacking FTO. Interestingly, FTO-mediated demethylation of snRNAs can be inhibited by specific intracellular metabolites, suggesting a model in which changes in metabolism may affect pre-mRNA splicing by altering the cap proximal methylation status of snRNAs. To date, no demethylases are known to target internal snRNA modifications, possibly indicating that the protein-rich environment of (partially) assembled snRNPs largely impedes access of demethylation enzymes to potential target sites.


References

Hari, R., & Parthasarathy, S. (2018). Prediction of Coding and Non-Coding RNA. Reference Module in Life Sciences. doi:10.1016/b978-0-12-809633-8.20099-x

Buratti, Emanuele. (2013). Small Nuclear RNA. 10.1016/B978-0-12-374984-0.01437-6.

https://www.frontiersin.org/articles/10.3389/fgene.2021.652129/full

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