Prp8

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Crystal Structure of Prp8
Crystal Structure of Prp8.png
Prp8 Crystal Structure
Identifiers
SymbolPRP8
Alt. symbolsUSA2, DBF3, DNA39, RNA8, SLT21
PDB 4I43
UniProt P33334
Search for
Structures Swiss-model
Domains InterPro

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. [1]

Contents

History

The systematic name for the PRP8 protein is YHR165C. Prp8 protein is coded by a single gene in humans with 42 exons. The size of Prp8 ranges between 230 and 280 kDa depending on the organism. The sequence coding for the Prp8 protein is highly conserved between eukaryotic organisms, with a 61% identity match between humans and yeast in amino acid sequence. [1] The Prp8 gene is located on chromosome VIII in yeast and chromosome 17 in humans.

Role in splicing

The splicing mechanism with Prp8 indicated in black. Slide2-splicing.jpg
The splicing mechanism with Prp8 indicated in black.

Pre-mRNA splicing involves two trans-esterification reactions and attacks by hydroxyl groups within the spliceosome. In these reactions, spliceosomal intron removal is catalyzed by the spliceosome using the same mechanism as Group II introns. [2] There are five key small nuclear RNA-protein complexes (snRNP) involved in this process. All of the snRNPs together contribute about 50 proteins to the core spliceosome. [2] The Prp8 gene encodes for a protein that is a central part of the U5 snRNP and the U5-U4/U6 tri-snRNP. The U5-U4/U6 tri-snRNP is involved with Complex B, the pre-catalytic spliceosome, where the U5 snRNP binds to exons at the 5’ end of the mRNA before shifting to introns. The U5 snRNP is involved with Complex C, the catalytic spliceosome, where the U5 snRNP binds to an exon at the 3’ splice site and the lariat loop forms. The U5 snRNP is also involved with Complex C*, the post-catalytic spliceosome, where it remains bound to the lariat before the spliced RNA is released and the snRNPs are recycled.

Common research methods for studying the structure and functions of Prp8 are co-immunoprecipitation and Western blot analysis. The structure of Prp8 includes a RNA recognition motif, a MPN / JAB ubiquitin-binding domain near the C-terminus, and a nuclear localization signal (NLS) that tags the protein to be moved to the cell nucleus. [3] The crystal structure of Prp8 protein (residues 885–2413) reveals tightly associated domains that resemble an intron reverse transcriptase and a type II restriction endonuclease. This implies that Prp8 might play roles similar to both the creation of cDNA and in cutting the DNA during splicing.

Labeled crystal structure of Prp8 bonded to Aar2. Slide1-structure.jpg
Labeled crystal structure of Prp8 bonded to Aar2.

Prp8 is also more involved with maintaining proper conformation of the bound RNA cofactors and substrates of the splicing reaction. Prp8, along with two other U5 snRNP proteins, helps to activate the spliceosome and form its catalytic active center. It has been proposed that GTP hydrolysis results in a rearrangement of Prp8 that releases the U1 and U4 snRNPs and is responsible for this activation of the catalytic core of the spliceosome. [4] Prp8 performs a scaffold-like function in the spliceosome and holds onto many of the interacting substrates and subunits. It has been cross-linked at both the 3’ and 5’ splice sites in mRNA. [5] Due to these structural elements, it has been assumed that Prp8 may have evolved from inactivated retroelements of reverse transcriptases, [6] with the snRNPs replacing the catalytic domains of self-splicing ancestors. [2]

Mutation and disease

Deficiencies

Prp8 mutation has been linked to the human disease Retinitis Pigmentosa causing vision loss, especially progressing into adulthood. This autosomal dominant affliction results with degeneration of the photoreceptors of the retina of the eye. This disorder is caused by mutations in the C-terminus. Retinitis Pigmentosa results from nine missense mutations in the last exon of the mature mRNA result with changes in seven highly conserved amino acids. Studies in yeast indicate that mutation of the C-terminus affects interactions with Brr2p, a helicase responsible for necessary function for the unwinding of the U1 snRNA/5’SS and U4/U6 RNA helices.

Phenotype mutations of Prp8 across species

Caenorhabditis elegans Prp8 has been linked to reproduction and development. RNAi, or RNA Interference, was used to knockout Prp8. This resulted in a high level of sterility, a clear body, and protruding vulva, all phenotypical expressions linked to reproduction and development. [7] [8] Mouse Prp8 mutation has resulted in Retinitis Pigmentosa (see above). [9] Yeast Prp8 mutation results in a U5 snRNP maturation defect. [10] The U5 snRNAP component of the splicesosome is necessary to bind to the 5' and 3' exons during pre-mRNA splicing. Mutations with this subunit correlate to reduced or inaccurate editing of RNA. In severe cases, mutations in Prp8 can lead to cell death.

See also

Related Research Articles

<span class="mw-page-title-main">RNA splicing</span> Process in molecular biology

RNA splicing is a process in molecular biology where a newly-made precursor messenger RNA (pre-mRNA) transcript is transformed into a mature messenger RNA (mRNA). It works by removing all the introns and splicing back together exons. For nuclear-encoded genes, splicing occurs in the nucleus either during or immediately after transcription. For those eukaryotic genes that contain introns, splicing is usually needed to create an mRNA molecule that can be translated into protein. For many eukaryotic introns, splicing occurs in a series of reactions which are catalyzed by the spliceosome, a complex of small nuclear ribonucleoproteins (snRNPs). There exist self-splicing introns, that is, ribozymes that can catalyze their own excision from their parent RNA molecule. The process of transcription, splicing and translation is called gene expression, the central dogma of molecular biology.

<span class="mw-page-title-main">Spliceosome</span> 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. Small nuclear RNA (snRNA) molecules bind to specific proteins to form a small nuclear ribonucleoprotein complex, which in turn combines with other snRNPs to form a large ribonucleoprotein complex called a spliceosome. 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.

<span class="mw-page-title-main">Minor spliceosome</span>

The minor spliceosome is a ribonucleoprotein complex that catalyses the removal (splicing) of an atypical class of spliceosomal introns (U12-type) from messenger RNAs in some clades of eukaryotes. 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.

<span class="mw-page-title-main">Group II intron</span> 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 insert into DNA sites has been exploited as a tool for biotechnology. For example, group II introns can be modified to make site-specific genome insertions and deliver cargo DNA such as reporter genes or lox sites

<span class="mw-page-title-main">U2 spliceosomal RNA</span>

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. 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. 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. 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.

<span class="mw-page-title-main">U4 spliceosomal RNA</span> 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.

<span class="mw-page-title-main">U5 spliceosomal RNA</span>

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.

<span class="mw-page-title-main">U6 spliceosomal RNA</span> Small nuclear RNA component of the spliceosome

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.

<span class="mw-page-title-main">PRPF8</span> Protein-coding gene in the species Homo sapiens

Pre-mRNA-processing-splicing factor 8 is a protein that in humans is encoded by the PRPF8 gene.

<span class="mw-page-title-main">PRPF31</span> Protein-coding gene in the species Homo sapiens

PRP31 pre-mRNA processing factor 31 homolog , also known as PRPF31, is a protein which in humans is encoded by the PRPF31 gene.

<span class="mw-page-title-main">PRPF3</span> Protein-coding gene in the species Homo sapiens

U4/U6 small nuclear ribonucleoprotein Prp3 is a protein that in humans is encoded by the PRPF3 gene.

<span class="mw-page-title-main">PRPF6</span> Protein-coding gene in the species Homo sapiens

Pre-mRNA-processing factor 6 is a protein that in humans is encoded by the PRPF6 gene.

<span class="mw-page-title-main">PRPF4</span> Protein-coding gene in the species Homo sapiens

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]

<span class="mw-page-title-main">RP9</span> Protein-coding gene in humans

Retinitis pigmentosa 9 (autosomal dominant), also known as RP9 or PAP-1, is a protein which in humans is encoded by the RP9 gene.

<span class="mw-page-title-main">DDX23</span> Protein-coding gene in humans

Probable ATP-dependent RNA helicase DDX23 is an enzyme that in humans is encoded by the DDX23 gene.

<span class="mw-page-title-main">Prp24</span>

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.

Christine Guthrie (1945-2022) was an American yeast geneticist and American Cancer Society Research Professor of Genetics at University of California San Francisco. She showed that yeast have small nuclear RNAs (snRNAs) involved in splicing pre-messenger RNA into messenger RNA in eukaryotic cells. Guthrie cloned and sequenced the genes for yeast snRNA and established the role of base pairing between the snRNAs and their target sequences at each step in the removal of an intron. She also identified proteins that formed part of the spliceosome complex with the snRNAs. Elected to the National Academy of Sciences in 1993, Guthrie edited Guide to Yeast Genetics and Molecular Biology, an influential methods series for many years.

<span class="mw-page-title-main">Kiyoshi Nagai</span> Japanese structural biologist (1949–2019)

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

  1. 1 2 Grainger RJ, Beggs JD (May 2005). "Prp8 protein: at the heart of the spliceosome". RNA. 11 (5): 533–57. doi:10.1261/rna.2220705. PMC   1370742 . PMID   15840809.
  2. 1 2 3 Cox DL, Nelson MM (2008). Lehninger Principles of Biochemistry (5th ed.). New York: W.H. Freeman. pp.  1037–1039. ISBN   9780716771081.
  3. ↑ Bellare P, Kutach AK, Rines AK, Guthrie C, Sontheimer EJ (February 2006). "Ubiquitin binding by a variant Jab1/MPN domain in the essential pre-mRNA splicing factor Prp8p". RNA. 12 (2): 292–302. doi:10.1261/rna.2152306. PMC   1370909 . PMID   16428608.
  4. ↑ Strauss, E.J.; Ben-Yehuda, S.; Umen, J.G.; Wiesner, S.; Brenner, T.J. "PRP8 - U4/U6-U5 snRNP complex component PRP8". WikiGenes. Collaborative Publishing. Retrieved 2 November 2015.
  5. ↑ Galej WP, Oubridge C, Newman AJ, Nagai K (January 2013). "Crystal structure of Prp8 reveals active site cavity of the spliceosome". Nature. 493 (7434): 638–43. Bibcode:2013Natur.493..638G. doi:10.1038/nature11843. PMC   3672837 . PMID   23354046.
  6. ↑ Dlakić M, Mushegian A (May 2011). "Prp8, the pivotal protein of the spliceosomal catalytic center, evolved from a retroelement-encoded reverse transcriptase". RNA. 17 (5): 799–808. doi:10.1261/rna.2396011. PMC   3078730 . PMID   21441348.
  7. ↑ Gönczy P, Echeverri C, Oegema K, Coulson A, Jones SJ, Copley RR, Duperon J, Oegema J, Brehm M, Cassin E, Hannak E, Kirkham M, Pichler S, Flohrs K, Goessen A, Leidel S, Alleaume AM, Martin C, Ozlü N, Bork P, Hyman AA (November 2000). "Functional genomic analysis of cell division in C. elegans using RNAi of genes on chromosome III". Nature. 408 (6810): 331–6. Bibcode:2000Natur.408..331G. doi:10.1038/35042526. PMID   11099034. S2CID   4364278.
  8. ↑ McKie AB, McHale JC, Keen TJ, Tarttelin EE, Goliath R, van Lith-Verhoeven JJ, Greenberg J, Ramesar RS, Hoyng CB, Cremers FP, Mackey DA, Bhattacharya SS, Bird AC, Markham AF, Inglehearn CF (July 2001). "Mutations in the pre-mRNA splicing factor gene PRPC8 in autosomal dominant retinitis pigmentosa (RP13)". Human Molecular Genetics. 10 (15): 1555–62. doi: 10.1093/hmg/10.15.1555 . PMID   11468273.
  9. ↑ Graziotto JJ, Farkas MH, Bujakowska K, Deramaudt BM, Zhang Q, Nandrot EF, Inglehearn CF, Bhattacharya SS, Pierce EA (January 2011). "Three gene-targeted mouse models of RNA splicing factor RP show late-onset RPE and retinal degeneration". Investigative Ophthalmology & Visual Science. 52 (1): 190–8. doi:10.1167/iovs.10-5194. PMC   3053274 . PMID   20811066.
  10. ↑ Boon KL, Grainger RJ, Ehsani P, Barrass JD, Auchynnikava T, Inglehearn CF, Beggs JD (November 2007). "prp8 mutations that cause human retinitis pigmentosa lead to a U5 snRNP maturation defect in yeast". Nature Structural & Molecular Biology. 14 (11): 1077–83. doi:10.1038/nsmb1303. PMC   2584834 . PMID   17934474.
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