U2 spliceosomal RNA

Last updated
U2 spliceosomal RNA
RF00004.jpg
Predicted secondary structure and sequence conservation of U2
Identifiers
SymbolU2
Rfam RF00004
Other data
RNA type Gene; snRNA; splicing
Domain(s) Eukaryota
GO GO:0000370 GO:0045131 GO:0005686
SO SO:0000392
PDB structures PDBe

U2 spliceosomal snRNAs are a species of small nuclear RNA (snRNA) molecules found in the major spliceosomal (Sm) machinery of virtually all eukaryotic organisms. In vivo, U2 snRNA along with its associated polypeptides assemble to produce the U2 small nuclear ribonucleoprotein (snRNP), an essential component of the major spliceosomal complex. [1] The major spliceosomal-splicing pathway is occasionally referred to as U2 dependent, based on a class of Sm intron—found in mRNA primary transcripts—that are recognized exclusively by the U2 snRNP during early stages of spliceosomal assembly. [2] In addition to U2 dependent intron recognition, U2 snRNA has been theorized to serve a catalytic role in the chemistry of pre-RNA splicing as well. [3] [4] Similar to ribosomal RNAs (rRNAs), Sm snRNAs must mediate both RNA:RNA and RNA:protein contacts and hence have evolved specialized, highly conserved, primary and secondary structural elements to facilitate these types of interactions. [5] [6]

Contents

Shortly after the discovery that mRNA primary transcripts contain long, non-coding intervening sequences (introns) by Sharp and Roberts, [7] [8] Joan Steitz began work to characterize the biochemical mechanism of intron excision. [9] The curious observation that a sequence found in the 5´ region of the U1 snRNA exhibited extensive base pairing complementarity with conserved sequences across 5´ splice junctions in hnRNA transcripts prompted speculation that certain snRNAs may be involved in recognizing splice site boundaries through RNA:RNA contacts. [9] Only recently have atomic crystal structures revealed demonstrably that the original conjecture was indeed correct, even if the complexity of these interactions were not fully realized at the time. [5] [6] [10]

U2 snRNA Recognition Elements

In Saccharomyces cerevisiae the U2 snRNA is associated with 18 polypeptides, seven of which are structural proteins common to all Sm class snRNPs. [11] These non-specific structural proteins associate with Sm snRNAs through a highly conserved recognition sequence (AUnG,n = 4-6) located within the RNA called Sm-binding sites. [12] Two other proteins, A´ and B´´, are U2-specific and require structural elements unique to U2 snRNA—specifically two 3´ stem loops—for snRNP assembly. [11] The three-subunit SF3a and six-subunit SF3b protein complexes also associate with the U2 snRNA. [13]

U2 snRNA is implicated in intron recognition through a 7-12 nucleotide sequence between 18-40 nucleotides upstream of the 3´ splice site known as the branch point sequence (BPS). [1] [2] In yeast, the consensus BPS is 7 nucleotide residues in length and the complementary recognition sequence within the U2 snRNA is 6 nucleotides. Duplex formation between these two sequences results in bulging of a conserved adenosine residue at position 5 of the BPS. The bulged adenosine residue adopts a C3´-endo conformation [14] that with the help of splicing factors Cwc25, Yju2 and Isy1 aligns a 2´ OH for an inline attack of a phosphorus atom at the 5´ splice site. [15] Nucleophilic attack initiates the first of two successive transesterification reactions that splices out the intron—through an unusual 2´-5´-3´ linked lariat intermediate—where the second transesterification involves ligation of the two flanking exons.

Primary and Secondary Structure

Although the sequence length of U2 snRNAs can vary by up to an order of magnitude across all eukaryotic organisms, all U2 snRNAs contain many phylogenetically constant regions particularly within the first 80 nucleotides downstream of the 5´ end where 85% of the positions are conserved. [16] Moreover, several secondary structural elements are also conserved including stem loops I, II, III, IV, and some of the single stranded regions linking these domains. [16] [17] Stem loop II in yeast U2 snRNA, contains an unusual sheared GA base pair leading into a characteristic U-turn loop motif that shares a geometric conformation similar to that of tRNA anti-codon loops. [5] All U2 snRNAs possess a terminal stem loop (IV) with a 10-16 base pair helix and a conserved 11 nucleotide loop with the consensus sequence 5´-UYGCANUURYN-3´. [16]

U2 snRNAs are the most extensively modified of all the small nuclear RNAs. [18] While the exact locations of these post-transcriptional modifications can vary from organism to organism, emerging evidence suggests there is a strong correlation between U2 snRNA modification and biological function. [18] Modifications include the conversion of some uridine residues to pseudouridine, 2´-O-methylation, nucleobase methylation, and conversion of 5´-monomethylated guanosine cap to a 2,2,7-trimethylated guanosine cap. [18] Many of these modifications reside in a 27-nucleotide region on the 5´ end of the molecule. [18]

Conformational Dynamics

The spliceosome is a dynamic molecular machine that undergoes several conformational rearrangements throughout assembly and splicing. Although many of the biochemical details surrounding spliceosomal rearrangements remains unclear, recent studies have visualized the formation of a critical folding complex between U2 and U6 snRNAs immediately proceeding the first step of the splicing reaction. [19] [6] This folding event facilitates the formation of a four-helix junction, which is believed to provide scaffolding for the critical components of the active site including aligning the 5´ splice site with the branch point adenosine for inline attack by the 2´ OH and coordinating two Mg2+ ions to stabilize negative charge formation in the proceeding steps. [19]

Evolutionary Origins

A notable characteristic of the U2-U6 fold is its structural similarity to that of domain V in self-splicing group II introns. [6] [3] The AGC triad found in U6 snRNA is conserved in group II introns and has been found to favor the same tertiary stacking interactions as well. [6] The formation of a GU wobble pair early in the U2-U6 folding event is also observed in the formation of the catalytic core of group II introns. [19] Finally, it is likely the spliceosome utilizes the same two-metal ion mechanism as group II introns given the structural conservation of metal binding sites found within the U2-U6 fold. [3] The extent of both secondary and tertiary structure conservation between group II introns and the U2-U6 fold in the active site of the spliceosome strongly suggests both group II introns and the spliceosome share a common evolutionary origin.

See also

Related Research Articles

An intron is any nucleotide sequence within a gene that is removed by RNA splicing during maturation of the final RNA product. In other words, introns are non-coding regions of an RNA transcript, or the DNA encoding it, that are eliminated by splicing before translation. The word intron is derived from the term intragenic region, i.e. a region inside a gene. The term intron refers to both the DNA sequence within a gene and the corresponding sequence in RNA transcripts. Sequences that are joined together in the final mature RNA after RNA splicing are exons.

RNA splicing Processing primary RNA to remove intron sequences and join the remaining exon sections

RNA splicing, in molecular biology, is a form of RNA processing in which a newly made precursor messenger RNA (pre-mRNA) transcript is transformed into a mature messenger RNA (mRNA). During splicing, introns are removed and exons are joined together. For nuclear-encoded genes, splicing takes place within the nucleus either during or immediately after transcription. For those eukaryotic genes that contain introns, splicing is usually required in order to create an mRNA molecule that can be translated into protein. For many eukaryotic introns, splicing is carried out in a series of reactions which are catalyzed by the spliceosome, a complex of small nuclear ribonucleoproteins (snRNPs). Self-splicing introns, or ribozymes capable of catalyzing their own excision from their parent RNA molecule, also exist.

Spliceosome Molecular machine that removes intron RNA from the primary transcript

A spliceosome is a large ribonucleoprotein (RNP) complex found primarily within the nucleus of eukaryotic cells. The spliceosome is assembled from small nuclear RNAs (snRNA) and numerous proteins. The spliceosome removes introns from a transcribed pre-mRNA, a type of primary transcript. This process is generally referred to as splicing. An analogy is a film editor, who selectively cuts out irrelevant or incorrect material from the initial film and sends the cleaned-up version to the director for the final cut.

snRNPs, or small nuclear ribonucleoproteins, are RNA-protein complexes that combine with unmodified pre-mRNA and various other proteins to form a spliceosome, a large RNA-protein molecular complex upon which splicing of pre-mRNA occurs. The action of snRNPs is essential to the removal of introns from pre-mRNA, a critical aspect of post-transcriptional modification of RNA, occurring only in the nucleus of eukaryotic cells. Additionally, U7 snRNP is not involved in splicing at all, as U7 snRNP is responsible for processing the 3′ stem-loop of histone pre-mRNA.

Small nuclear RNA (snRNA) is a class of small RNA molecules that are found within the splicing speckles and Cajal bodies of the cell nucleus in eukaryotic cells. The length of an average snRNA is approximately 150 nucleotides. They are transcribed by either RNA polymerase II or RNA polymerase III. Their primary function is in the processing of pre-messenger RNA (hnRNA) in the nucleus. They have also been shown to aid in the regulation of transcription factors or RNA polymerase II, and maintaining the telomeres.

Minor spliceosome

The minor spliceosome is a ribonucleoprotein complex that catalyses the removal (splicing) of an atypical class of spliceosomal introns (U12-type) from eukaryotic messenger RNAs in plants, insects, vertebrates and some fungi. This process is called noncanonical splicing, as opposed to U2-dependent canonical splicing. U12-type introns represent less than 1% of all introns in human cells. However they are found in genes performing essential cellular functions.

Group II intron Class of self-catalyzing ribozymes

Group II introns are a large class of self-catalytic ribozymes and mobile genetic elements found within the genes of all three domains of life. Ribozyme activity can occur under high-salt conditions in vitro. However, assistance from proteins is required for in vivo splicing. In contrast to group I introns, intron excision occurs in the absence of GTP and involves the formation of a lariat, with an A-residue branchpoint strongly resembling that found in lariats formed during splicing of nuclear pre-mRNA. It is hypothesized that pre-mRNA splicing may have evolved from group II introns, due to the similar catalytic mechanism as well as the structural similarity of the Group II Domain V substructure to the U6/U2 extended snRNA. Finally, their ability to site-specifically mobilize to new DNA sites has been exploited as a tool for biotechnology.

LSm

In molecular biology, LSm proteins are a family of RNA-binding proteins found in virtually every cellular organism. LSm is a contraction of 'like Sm', because the first identified members of the LSm protein family were the Sm proteins. LSm proteins are defined by a characteristic three-dimensional structure and their assembly into rings of six or seven individual LSm protein molecules, and play a large number of various roles in mRNA processing and regulation.

U11 spliceosomal RNA

The U11 snRNA is an important non-coding RNA in the minor spliceosome protein complex, which activates the alternative splicing mechanism. The minor spliceosome is associated with similar protein components as the major spliceosome. It uses U11 snRNA to recognize the 5' splice site while U12 snRNA binds to the branchpoint to recognize the 3' splice site.

U1 spliceosomal RNA

U1 spliceosomal RNA is the small nuclear RNA (snRNA) component of U1 snRNP, an RNA-protein complex that combines with other snRNPs, unmodified pre-mRNA, and various other proteins to assemble a spliceosome, a large RNA-protein molecular complex upon which splicing of pre-mRNA occurs. Splicing, or the removal of introns, is a major aspect of post-transcriptional modification, and takes place only in the nucleus of eukaryotes.

U4 spliceosomal RNA Non-coding RNA component of the spliceosome

The U4 small nuclear Ribo-Nucleic Acid is a non-coding RNA component of the major or U2-dependent spliceosome – a eukaryotic molecular machine involved in the splicing of pre-messenger RNA (pre-mRNA). It forms a duplex with U6, and with each splicing round, it is displaced from the U6 snRNA in an ATP-dependent manner, allowing U6 to re-fold and create the active site for splicing catalysis. A recycling process involving protein Brr2 releases U4 from U6, while protein Prp24 re-anneals U4 and U6. The crystal structure of a 5′ stem-loop of U4 in complex with a binding protein has been solved.

U5 spliceosomal RNA

U5 snRNA is a small nuclear RNA (snRNA) that participates in RNA splicing as a component of the spliceosome. It forms the U5 snRNP by associating with several proteins including Prp8 - the largest and most conserved protein in the spliceosome, Brr2 - a helicase required for spliceosome activation, Snu114, and the 7 Sm proteins. U5 snRNA forms a coaxially-stacked series of helices that project into the active site of the spliceosome. Loop 1, which caps this series of helices, forms 4-5 base pairs with the 5'-exon during the two chemical reactions of splicing. This interaction appears to be especially important during step two of splicing, exon ligation.

U6 spliceosomal RNA

U6 snRNA is the non-coding small nuclear RNA (snRNA) component of U6 snRNP, an RNA-protein complex that combines with other snRNPs, unmodified pre-mRNA, and various other proteins to assemble a spliceosome, a large RNA-protein molecular complex that catalyzes the excision of introns from pre-mRNA. Splicing, or the removal of introns, is a major aspect of post-transcriptional modification and takes place only in the nucleus of eukaryotes.

SF3B2

Splicing factor 3B subunit 2 is a protein that in humans is encoded by the SF3B2 gene.

SNRPB2

U2 small nuclear ribonucleoprotein B is a protein that in humans is encoded by the SNRPB2 gene.

PRPF4

U4/U6 small nuclear ribonucleoprotein Prp4 is a protein that in humans is encoded by the PRPF4 gene. The removal of introns from nuclear pre-mRNAs occurs on complexes called spliceosomes, which are made up of 4 small nuclear ribonucleoprotein (snRNP) particles and an undefined number of transiently associated splicing factors. PRPF4 is 1 of several proteins that associate with U4 and U6 snRNPs.[supplied by OMIM]

SmY RNA

SmY ribonucleic acids are a family of small nuclear RNAs found in some species of nematode worms. They are thought to be involved in mRNA trans-splicing.

Prp24

Prp24 is a protein part of the pre-messenger RNA splicing process and aids the binding of U6 snRNA to U4 snRNA during the formation of spliceosomes. Found in eukaryotes from yeast to E. coli, fungi, and humans, Prp24 was initially discovered to be an important element of RNA splicing in 1989. Mutations in Prp24 were later discovered in 1991 to suppress mutations in U4 that resulted in cold-sensitive strains of yeast, indicating its involvement in the reformation of the U4/U6 duplex after the catalytic steps of splicing.

Prp8

Prp8 refers to both the Prp8 protein and Prp8 gene. Prp8's name originates from its involvement in pre-mRNA processing. The Prp8 protein is a large, highly conserved, and unique protein that resides in the catalytic core of the spliceosome and has been found to have a central role in molecular rearrangements that occur there. Prp8 protein is a major central component of the catalytic core in the spliceosome, and the spliceosome is responsible for splicing of precursor mRNA that contains introns and exons. Unexpressed introns are removed by the spliceosome complex in order to create a more concise mRNA transcript. Splicing is just one of many different post-transcriptional modifications that mRNA must undergo before translation. Prp8 has also been hypothesized to be a cofactor in RNA catalysis.

Kiyoshi Nagai Japanese structural biologist

Kiyoshi Nagai was a Japanese structural biologist at the MRC Laboratory of Molecular Biology Cambridge, UK. He was known for his work on the mechanism of RNA splicing and structures of the spliceosome.

References

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  4. ↑ Shi Y (August 2017). "The Spliceosome: A Protein-Directed Metalloribozyme". Journal of Molecular Biology. 429 (17): 2640–2653. doi: 10.1016/j.jmb.2017.07.010 . PMID   28733144.
  5. 1 2 3 Stallings, Sarah C; Moore, Peter B (1997). "The structure of an essential splicing element: stem loop IIa from yeast U2 snRNA". Structure. 5 (9): 1173–1185. doi: 10.1016/s0969-2126(97)00268-2 . ISSN   0969-2126. PMID   9331416.
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  7. ↑ Berget SM, Moore C, Sharp PA (August 1977). "Spliced segments at the 5' terminus of adenovirus 2 late mRNA". Proceedings of the National Academy of Sciences of the United States of America. 74 (8): 3171–5. Bibcode:1977PNAS...74.3171B. doi:10.1073/pnas.74.8.3171. PMC   431482 . PMID   269380.
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  9. 1 2 Lerner MR, Boyle JA, Mount SM, Wolin SL, Steitz JA (January 1980). "Are snRNPs involved in splicing?". Nature. 283 (5743): 220–4. Bibcode:1980Natur.283..220L. doi:10.1038/283220a0. PMID   7350545. S2CID   4266714.
  10. ↑ Perriman R, Ares M (May 2010). "Invariant U2 snRNA nucleotides form a stem loop to recognize the intron early in splicing". Molecular Cell. 38 (3): 416–27. doi:10.1016/j.molcel.2010.02.036. PMC   2872779 . PMID   20471947.
  11. 1 2 Pan ZQ, Prives C (December 1989). "U2 snRNA sequences that bind U2-specific proteins are dispensable for the function of U2 snRNP in splicing". Genes & Development. 3 (12A): 1887–98. doi: 10.1101/gad.3.12a.1887 . PMID   2559872.
  12. ↑ Mattaj IW, Habets WJ, van Venrooij WJ (May 1986). "Monospecific antibodies reveal details of U2 snRNP structure and interaction between U1 and U2 snRNPs". The EMBO Journal. 5 (5): 997–1002. doi:10.1002/j.1460-2075.1986.tb04314.x. PMC   1166893 . PMID   2941274.
  13. ↑ Dziembowski, Andrzej; Ventura, Ana-Paula; Rutz, Berthold; Caspary, Friederike; Faux, Céline; Halgand, Frédéric; Laprévote, Olivier; Séraphin, Bertrand (2004-12-08). "Proteomic analysis identifies a new complex required for nuclear pre-mRNA retention and splicing". The EMBO Journal. 23 (24): 4847–4856. doi:10.1038/sj.emboj.7600482. ISSN   0261-4189. PMC   535094 . PMID   15565172.
  14. ↑ Berglund JA, Rosbash M, Schultz SC (May 2001). "Crystal structure of a model branchpoint-U2 snRNA duplex containing bulged adenosines". RNA. 7 (5): 682–91. doi:10.1017/S1355838201002187. PMC   1370120 . PMID   11350032.
  15. ↑ Galej, Wojciech P.; Wilkinson, Max E.; Fica, Sebastian M.; Oubridge, Chris; Newman, Andrew J.; Nagai, Kiyoshi (8 September 2016). "Cryo-EM structure of the spliceosome immediately after branching". Nature. 537 (7619): 197–201. Bibcode:2016Natur.537..197G. doi:10.1038/nature19316. ISSN   1476-4687. PMC   5156311 . PMID   27459055.
  16. 1 2 3 Guthrie C, Patterson B (1988). "Spliceosomal snRNAs". Annual Review of Genetics. 22: 387–419. doi:10.1146/annurev.ge.22.120188.002131. PMID   2977088.
  17. ↑ Krämer A (August 1987). "Analysis of RNase-A-resistant regions of adenovirus 2 major late precursor-mRNA in splicing extracts reveals an ordered interaction of nuclear components with the substrate RNA". Journal of Molecular Biology. 196 (3): 559–73. doi:10.1016/0022-2836(87)90032-5. PMID   3681967.
  18. 1 2 3 4 Yu YT, Shu MD, Steitz JA (October 1998). "Modifications of U2 snRNA are required for snRNP assembly and pre-mRNA splicing". The EMBO Journal. 17 (19): 5783–95. doi:10.1093/emboj/17.19.5783. PMC   1170906 . PMID   9755178.
  19. 1 2 3 Sashital DG, Cornilescu G, McManus CJ, Brow DA, Butcher SE (December 2004). "U2-U6 RNA folding reveals a group II intron-like domain and a four-helix junction". Nature Structural & Molecular Biology. 11 (12): 1237–42. doi:10.1038/nsmb863. PMID   15543154. S2CID   35368436.

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