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U5 snRNP

 

Discovery

When the U5 RNA was first purified, it was known as the “5S RNAIII” (Bush et AL).  Suk Ro-Choi, Reddy, Henning and Busch first found 5S RNA III in the “Novikoff hepatoma ascites cell nuclei in 1971”. (Suk Ro-Choi, 1971) It was isolated by chromatography on DEAE Sephadex columns and the oligonucleotides of 5S RNA III were produced from Rnase digestion.   In 1971, the function was not known. The structure was found to be Uridine rich.

In 1985, Benoit Chabot, Douglas Black, David LeMaster, and Joan Stietz speculated that the U5 protein might be involved in splicing.  A “component in mammalian splicing extracts” interacts with the “3’ splicie site of the first intron of human beta-globin pre-mRNA. The group of scientists identified the U5 protein by examining that the “binding activity was resistant to micrococcal nuclease,” in which the only snRNP’s that contained resistance to this nuclease was the U5 RNA. (Chabot, Black, LeMaster, and Stietz, 1985).

Also in 1985, Paula Grabowski and Phillip Sharp using “affinity chromatography based on the high specificity of the biotin-streptavidin interaction identified the U2, U5 and the U4+U6 small nuclear ribonucleoprotein particles in the 35s spliceosome.” (Grabowski and Sharp, 1985).

 

Function

The U5 snRNA is thought to contribute functionally during the two transeterification reactions. The U5 snRNA carries an “invairant seqence of nine nucleotides displayed in an 11 nucleotide loop. (Newman, 1997). The U5 protein forms the B1 complex with U4/U5/U6.  In yeast the U5 loop interacts with exon sequences at the 5’ and 3’ splice sites. The U5 protein carries binding sites for the 5’ and 3’ exons, which allows the spliceosome to “tether” the 5’ exon intermediate produced by the first catalytic step. The U5 protein then serves in alignment of the 5’ and 3’ exons involved in the second catalytic step.  That is, the U5 positions the exons for splicing (Newman, 1997).  The 3’ end of the first exon and the 5’ end of the second exon are binded.

Picture of the U5 positioning near the exons for splicing:

http://www.emboj.org/cgi/content/full/16/19/5797/F1

The functionality of the U5 snRNP was performed by abalation of U5 snRNPs in yeast splicing extracts. It showed that in the absence of the U5 snRNPs, no splicing occurs and that spliceosome assembly is blocked.  Also it was found that the first catalytic step can occur without the U5 loop, but the second catalytic was inhibited without the U5 loop. 4-thio Crosslinking experimnets by Sootheimer and Steitz “provided evidence for the involment of U5 with the ends of exons during splicing” (Weaver, 1999 - text).  Steiz and Sootheimer also mapped the bases by reverse transcriptase and crosslinking, in which the reverse transcriptase would stop with cross-linking (Weaver, 1999 - text).

 

 

Figure: Human spliceosomal protein U5 (Reuter et al., 1999)

Further information about this structure may be obtained here:

http://www.rcsb.org/pdb/cgi/explore.cgi?pid=12342989381560&page=0&pdbId=1QGV

 

 

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