Spliceosome

A spliceosome is a complex of specialized RNA and protein subunits that removes introns from a transcribed pre-mRNA (hnRNA)segment. This process is generally referred to as splicing.

Composition

Each spliceosome is composed of five small nuclear RNA proteins, called snRNPs, (pronounced "snurps") and a range of non-snRNP associated protein factors.

The snRNPs that make up the nuclear spliceosome are named U1, U2, U4, U5, and U6, and participate in several RNA-RNA and RNA-protein interactions. The RNA component of the snRNP is rich in uridine (a variant of the uracil nucleotide).

The canonical assembly of the spliceosome occurs anew on each hnRNA. The hnRNA contains specific sequence elements that are recognized and utilized during spliceosome assembly. These include the 5' end splice, the branch point sequence, the polypyrimidine tract, and the 3' end splice site. The spliceosome catalyzes the removal of introns, and the ligation of the flanking exons.

Introns typically have a GU nucleotide sequence at the 5' end splice site, and an AG at the 3' end splice site. The 3' splice site can be further defined by a variable length of polypyrimidines, called the polypyrimidine tract (PPT), which serves the dual function of recruiting factors to the 3' splice site and possibly recruiting factors to the branch point sequence (BPS). The BPS contains the conserved Adenosine required for the first step of splicing.

A group of less abundant snRNPs, U11, U12, U4atac, and U6atac, together with U5, are subunits of the so-called minor spliceosome that splices a rare class of pre-mRNA introns, denoted U12-type. These snRNPs form the U12 spliceosome in the cytosolcite journal |author= König H, Matter N, Bader R, Thiele W, Müller F |title=Splicing Segregation: The Minor Spliceosome Acts outside the Nucleus and Controls Cell Proliferation |journal=Cell |volume=131 |issue=4 |pages=718–29 |year=2007 |pmid=18022366 |doi= 10.1016/j.cell.2007.09.043] .

New evidence derived from the first crystal structure of a group II intron suggests that the spliceosome is actually a ribozyme, and that it uses a two–metal ion mechanism for catalysis.cite journal |author= Toor N, Keating KS, Taylor SD, Pyle AM |title= Crystal structure of a self-spliced group II intron |journal= Science|volume=320 |issue=5872 |pages=77–82 |year=2008 |pmid=18388288 |doi= 10.1126/science.1153803]

Alternative splicing

Alternative splicing (the re-combination of different exons) is a major source of genetic diversity in eukaryotes. Splice variants have been used to account for the relatively small number of genes in the human genome. For years the estimate widely varied with top estimates reaching 100,000 genes,cite journal
author = Smaglik, P.
year = 2000
title = Researchers take a gamble on the human genome.
journal = Nature
volume = 405
issue = 6784
pages = 264
url = http://www.nature.com/nature/journal/v405/n6784/full/405264c0.html
doi = 10.1038/35012771
pmid = 10830930] but now, due to the Human Genome Project the figure is believed to be closer to 20,000 genes. One particular Drosophila gene (DSCAM) can be alternatively spliced into 38,000 different mRNA.cite journal
author = Schmucker, D.
coauthors = Clemens, J.C.; Shu, H.; Worby, C.A.; Xiao, J.; Muda, M.; Dixon, J.E.; Zipursky, S.L.
year = 2000
title = Drosophila Dscam Is an Axon Guidance Receptor Exhibiting Extraordinary Molecular Diversity
journal = Cell
volume = 101
issue = 6
pages = 671–684
pmid = 10892653

doi = 10.1016/S0092-8674(00)80878-8]

RNA splicing

In 1977, work by the Sharp and Roberts labs revealed that genes of higher organisms are "split" or present in several distinct segments along the DNA molecule.cite journal| title = Spliced segments at 5' terminus of adenovirus 2 late messenger-RNA
url = http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=269380
author = Berget, S. M., Moore, C. and Sharp, P. A.
date = 1977
journal = Proc. Natl. Acad. Sci. USA
volume = 74
pages = 3171–3175.
pmid = 269380
doi = 10.1073/pnas.74.8.3171] cite journal | author = Chow, L. T.
coauthors = Roberts, J. M., Lewis, J. B. and Broker, T. R.
title = A map of cytoplasmic RNA transcripts from lytic adenovirus type 2, determined by electron microscopy of RNA:DNA hybrids
date=1977
journal = Cell
volume = 11
pages = 819–836
doi = 10.1016/0092-8674(77)90294-X
pmid = 890740] The coding regions of the gene are separated by non-coding DNA that is not involved in protein expression. The split gene structure was found when adenoviral mRNAs were hybridized to endonuclease cleavage fragments of single stranded viral DNA. It was observed that the mRNAs of the mRNA-DNA hybrids contained 5' and 3' tails of non-hydrogen bonded regions. When larger fragments of viral DNAs were used, forked structures of looped out DNA were observed when hybridized to the viral mRNAs. It was realized that the looped out regions, the introns, are excised from the precursor mRNAs in a process Sharp named "splicing". The split gene structure was subsequently found to be common to most eukaryotic genes. Phillip Sharp and Richard J. Roberts were awarded the 1993 Nobel Prize in Physiology or Medicine for their discovery of introns and the splicing process.

pliceosome assembly

The model for formation of the spliceosome active site involves an ordered, stepwise assembly of discrete snRNP particles on the hnRNA substrate. The first recognition of hnRNAs involves U1 snRNP binding to the 5' end splice site of the hnRNA and other non-snRNP associated factors to form the commitment complex, or early (E) complex in mammals. [cite journal | journal=Molecular and Cell Biology | volume=12 | author = Jamison SF, Crow A, and Garcia-Blanco MA | date=1992 | title=The Spliceosome Assembly Pathway in Mammilian Extracts | pages=4279–4287 | pmid=1383687 | url=http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=1383687 ] [cite journal| journal= Cell | volume=59 | pages=349–358 | title=Identification of functional U1 snRNA pre-messenger RNA complexes committed to spliceosome assembly and splicing| author=Seraphin B. and Rosbash M. | date=1989 | pmid=2529976 | doi=10.1016/0092-8674(89)90296-1 ] The commitment complex is an ATP-independent complex that commits the hnRNA to the splicing pathway.cite journal |author=Legrain P, Seraphin B, Rosbash M |title=Early commitment of yeast pre-mRNA to the spliceosome pathway |journal=Mol. Cell. Biol. |volume=8 |issue=9 |pages=3755–60 |year=1988 |url=http://mcb.asm.org/cgi/reprint/8/9/3755 |pmid=3065622] U2 snRNP is recruited to the branch region through interactions with the E complex component U2AF (U2 snRNP auxiliary factor) and possibly U1 snRNP. In an ATP-dependent reaction, U2 snRNP becomes tightly associated with the branch point sequence (BPS) to form complex A. A duplex formed between U2 snRNA and the hnRNA branch region bulges out the branch adenosine specifying it as the nucleophile for the first transesterification. [cite journal
author = Query, C. C., M. J. Moore, and P. Sharp
date= 1994
title = Branch nucleophile selection in pre-mRNA splicing: evidence for the bulged duplex model
journal = Genes Devel.
volume = 8
pages = 587–597
url = http://www.genesdev.org/cgi/pmidlookup?view=long&pmid=7926752
doi=10.1101/gad.8.5.587
pmid=7926752]

The presence of a pseudouridine residue in U2 snRNA, nearly opposite of the branch site, results in an altered conformation of the RNA-RNA duplex upon the U2 snRNP binding. Specifically, the altered structure of the duplex induced by the pseudouridine places the 2' OH of the bulged adenosine in a favorable position for the first step of splicing. [cite journal
author = Newby M. I. and Greenbaum, N. L.
title=Sculpting of the spliceosomal branch site recognition motif by a conserved pseudouridine
journal = Nature Structural Biology
volume = 9
pages = 958–965
date = 2002
pmid = 12426583
doi = 10.1038/nsb873] The U4/U5/U6 tri-snRNP is recruited to the assembling spliceosome to form complex B, and following several rearrangements, complex C (the spliceosome) is activated for catalysis. [Citation|author = Burge, C.B., et al. |date = 1999 |chapter= Splicing precursors to mRNAs by the spliceosomes|editor=Gesteland, R.F., Cech, T.R., Atkins, J.F. |title=The RNA World|publisher=Cold Spring Harbor Lab. Press|pages=525–560] cite journal |author=Staley JP, Guthrie C |title=Mechanical devices of the spliceosome: motors, clocks, springs, and things |journal=Cell |volume=92 |issue=3 |pages=315–26 |year=1998 |pmid=9476892 |doi=10.1016/S0092-8674(00)80925-3] It is unclear how the triple snRNP is recruited to complex A, but this process may be mediated through protein-protein interactions and/or base pairing interactions between U2 snRNA and U6 snRNA.

The U5 snRNP interacts with sequences at the 5' and 3' splice sites via the invariant loop of U5 snRNAcite journal |author = Newman AJ, Teigelkamp S and Beggs JD | date = 1995 | title= snRNA interactions at 5' and 3' splice sites monitored by photoactivated crosslinking in yeast spliceosomes
journal = RNA
volume = 1
pages = 968–980
pmid=8548661
url = http://www.rnajournal.org/cgi/reprint/1/9/968] and U5 protein components interact with the 3' splice site region.cite journal |author=Chiara MD, Palandjian L, Feld Kramer R, Reed R |title=Evidence that U5 snRNP recognizes the 3' splice site for catalytic step II in mammals |journal=EMBO J. |volume=16 |issue=15 |pages=4746–59 |year=1997 |pmid=9303319 |doi=10.1093/emboj/16.15.4746 | url =http://www.nature.com/emboj/journal/v16/n15/abs/7590453a.html]

Upon recruitment of the triple snRNP, several RNA-RNA rearrangements precede the first catalytic step and further rearrangements occur in the catalytically active spliceosome. Several of the RNA-RNA interactions are mutually exclusive; however, it is not known what triggers these interactions, nor the order of these rearrangements. The first rearrangement is probably the displacement of U1 snRNP from the 5' splice site and formation of a U6 snRNA interaction. It is known that U1 snRNP is only weakly associated with fully formed spliceosomes [cite journal
author = Moore, M. J. and Sharp, P. A.
journal = Nature
volume = 365
pages = 364–368
date = 1993
title= Evidence for two active sites in the spliceosome provided by stereochemistry of pre-mRNA splicing
doi= 10.1038/365364a0
pmid=8397340] , and U1 snRNP is inhibitory to the formation of a U6-5' splice site interaction on a model of substrate oligonucleotide containing a short 5' exon and 5' splice site.cite journal |author=Konforti BB, Koziolkiewicz MJ, Konarska MM |title=Disruption of base pairing between the 5' splice site and the 5' end of U1 snRNA is required for spliceosome assembly |journal=Cell |volume=75 |issue=5 |pages=863–73 |year=1993 |pmid=8252623 |doi=10.1016/0092-8674(93)90531-T] Binding of U2 snRNP to the branch point sequence (BPS) is one example of an RNA-RNA interaction displacing a protein-RNA interaction. Upon recruitment of U2 snRNP, the branch binding protein SF1 in the commitment complex is displaced since the binding site of U2 snRNA and SF1 are mutually exclusive events.

Within the U2 snRNA, there are other mutually exclusive rearrangements that occur between competing conformations. For example, in the active form, stem loop IIa is favored; in the inactive form a mutually exclusive interaction between the loop and a downstream sequence predominates. It is unclear how U4 is displaced from U6 snRNAm, although RNA has been implicated in spliceosome assembly, and may function to unwind U4/U6 and promote the formation of a U2/U6 snRNA interaction. The interactions of U4/U6 stem loops I and II dissociate and the freed stem loop II region of U6 folds on itself to form an intramolecular stem loop and U4 is no longer required in further spliceosome assembly. The freed stem loop I region of U6 base pairs with U2 snRNA forming the U2/U6 helix I. However, the helix I structure is mutually exclusive with the 3' half of an internal 5' stem loop region of U2 snRNA.

References

*cite book |author=Alberts, Bruce "et al." |title=Essential cell biology |edition = Second edition |publisher=Garland Science, Taylor & Francis Group |location=New York and London |year=2004 |isbn=08-153-348-0X

*cite book |author=Alberts, Bruce "et al."|title=Molecular biology of the cell |publisher=Garland Science, Taylor and Francis Group |location=New York and London |year=2002 |edition=4th edition |chapter=6. How Cells Read the Genome: From DNA to Protein |isbn=0-8153-3218-1

External links

*cite journal |author=Nilsen T |title=The spliceosome: the most complex macromolecular machine in the cell? |journal=Bioessays |volume=25 |issue=12 |pages=1147–9 |year=2003 |pmid=14635248 |doi=10.1002/bies.10394
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