The network of protein-protein interactions within the human U4/U6.U5 tri-snRNP.
The human 25S U4/U6.U5 tri-snRNP is a major building block of the U2-type spliceosome and contains, in addition to the U4, U6, and U5 snRNAs, at least 30 distinct proteins. To learn more about the molecular architecture of the tri-snRNP, we have investigated interactions between tri-snRNP proteins using the yeast two-hybrid assay and in vitro binding assays, and, in addition, have identified distinct protein domains that are critical for the connectivity of this protein network in the human tri-snRNP. These studies revealed multiple interactions between distinct domains of the U5 proteins hPrp8, hBrr2 (a DExH/D-box helicase), and hSnu114 (a putative GTPase), which are key players in the catalytic activation of the spliceosome, during which the U4/U6 base-pairing interaction is disrupted and U4 is released from the spliceosome. Both the U5-specific, TPR/HAT-repeat-containing hPrp6 protein and the tri-snRNP-specific hSnu66 protein interact with several U5- and U4/U6-associated proteins, including hBrr2 and hPrp3, which contacts the U6 snRNA. Thus, both proteins are located at the interface between U5 and U4/U6 in the tri-snRNP complex, and likely play an important role in transmitting the activity of hBrr2 and hSnu114 in the U5 snRNP to the U4/U6 duplex during spliceosome activation. A more detailed analysis of these protein interactions revealed that different HAT repeats mediate interactions with specific hPrp6 partners. Taken together, data presented here provide a detailed picture of the network of protein interactions within the human tri-snRNP.
Liu
Sunbin
S
Department of Cellular Biochemistry, MPI of Biophysical Chemistry, Göttingen, Germany.
Rauhut
Reinhard
R
Vornlocher
Hans-Peter
HP
Lührmann
Reinhard
R
D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D019143:Evolution, Molecular; D017930:Genes, Reporter; D005982:Glutathione Transferase; D006801:Humans; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D012326:RNA Splicing; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D017414:Ribonucleoprotein, U4-U6 Small Nuclear / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics
D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D017414:Ribonucleoprotein, U4-U6 Small Nuclear; D005982:Glutathione Transferase