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U4 SnRNP

Discovery

Yeast U4 SnRNP was discovered in 1984 by Bringmann et al (Bringmann et al., 1984). The protein forms one part of a U4/U6 protein complex. By using affinity chromatography with 2,2,7-trimethyl-guanosine (m3G), which is present in the 5’-terminal caps of the SnRNAs, they saw that U4 SnRNP and U6 SnRNP were eluted together under high salt concentrations. Since U6 RNA lacks the m3G residue and does not react with anti-m3G, the fact that it eluted with U4 RNP on anti-3mG affinity columns indicates that either, the discrete SnRNPs U4 and U6 are intimately associated in nuclear extracts, or that both RNAs are organized in one ribonucleoprotein particle.

Scientists had difficulty in attaining purified U4 SnRNPs from yeast cells. However, Raghunathan and Guthri found that when they added Brr2 (a protein necessary for the dissasembly of U4 and U6 RNP’s) they attained purified U4 SnRNPs (Raghunathan et al., 1998).

In HeLa cells, nuclear extracts are subjected to several types of ion exchange chromatography at moderate ionic strength, electrophoresis on agarose gels, transfer of the particles on DEAE cellulose paper, and elution with ammonium chloride. They found the U4 and U6 SnRNPs contained U3 and U6 RNAs respectively and maintained their antigenicity as seen by their reaction with auto-antibodies from patients with lupus erythematosus (Pironcheva et al., 1988).

Function

After the U4/U6.U5 triple SnRNP adds to the spliceosome, the triple SnRNP escorts U6 to the spliceosome. U4 is base paired by 24 base pairs to U6 via stems I and II, and this interaction is disrupted as the spliceosome undergoes remodeling to be activated for catalysis. U4 leaves the stem II region of U6 by an ATP dependent mechanism. U6 then refolds to form an intramolecular stem loop which can base pair with SnRNA to form a U2/U6 helix.

The essential role of the U4/U6 SnRNP in mRNA splicing was suggested as a reuslt of inactivation experiments by Berget and Robbenson in 1986 (Berget et al., 1986). They degraded U4 SnRNA by a combination of ribonuclease H and an oligodeoxynucleotide complementary to sequences of U4 snRNA and found that splicing activity was inactivated in the absence of U4 SnRNA.

Interactions

In 1987, Chen and Abelson showed the sequence in which the spliceosome complex forms by fractionating splicing reaction mixtures on nondenaturing polyacrylamide gels and examining the complexes formed and the sequence in which the complexes form. They saw that snR14 (U4 SnRNA in mammals), snR7 (U5 snRNA in mammals), and snR6 (U6 SnRNA in mammals) formed a single large complex while the majority of snR6 was in a smaller complex. When ATP was added to the whole-cell extract, the large complex disappeared releasing snR7. They then saw that snR14 left the spliceosome and that when it did, there was a change in mobility, indicating a conformational change (Chen et al.,1987).

Figure: This is the human spliceosomal 15.5Kd protein bound to a U4 snRNA fragment (Vidovic et al., 2000)

For further information on this structure visit the following link:
http://www.rcsb.org/pdb/cgi/explore.cgi?pid=12342989381560&page=0&pdbId=1E7K

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