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. 2006 Jul;12(7):1418-30.
doi: 10.1261/rna.55406. Epub 2006 May 24.

The network of protein-protein interactions within the human U4/U6.U5 tri-snRNP

Sunbin Liu  1 , Reinhard Rauhut, Hans-Peter Vornlocher, Reinhard Lührmann
Affiliations

The network of protein-protein interactions within the human U4/U6.U5 tri-snRNP

Sunbin Liu et al. RNA. 2006 Jul.

Abstract

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.

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Figures

FIGURE 1.
FIGURE 1.
Two-hybrid interactions within the human tri-snRNP. All 29 full-length (F) proteins and protein fragments served as bait and prey in 29 × 29 Y2H combinations. Fragments are numbered as shown in Figure 2. Numbers adjacent to symbols indicate the highest tested 3-AT concentration (in mM) still supporting the interaction.
FIGURE 2.
FIGURE 2.
(A) Schematic representation of the hPrp8, hBrr2, and hSnu114 proteins and their fragments used in two-hybrid assays. (DExH) DExH box helicase domain; (HELICc) helicase superfamily C-terminal domain; (SEC63) domain of unknown function in Sec63p and other proteins; (EF-2_G) eukaryotic elongation factor 2 GTP-binding domain; (EF-2_II [IV, V]) eukaryotic elongation factor 2 domains II, IV, and V. (B) Observed interactions between the hPrp8, hBrr2, and hSnu114 proteins.
FIGURE 3.
FIGURE 3.
Interactions of the hPrp6 protein. (A) Summary of yeast two-hybrid and in vitro interactions involving hPrp6. (*) These interactions were identified previously (Makarova et al. 2002). (B) GST pull-downs performed with GST alone or GST-hPrp6 (as indicated) and in vitro translated c-Myc-tagged tagged hPrp3, hPrp4, hPrp31, hPrp6, hSnu66, fragment hPrp8–1, and fragment hBrr2–4 (indicated on the right). The input lane contains 10% of the total amount of in vitro translated protein added to each reaction. The input and bound proteins were analyzed by SDS-PAGE and visualized by autoradiography. (C) Co-IP experiments with purified His-tagged hDib1 and in vitro translated [35S]-methionine-labeled c-Myc-hPrp6 protein. His-tagged hDib1 was precipitated with anti-His antibodies, and co-precipitating proteins were again visualized by autoradiography following SDS-PAGE. A mock precipitation without His-tagged hDib1 is included as a negative control. The input lane represents 20% of the c-Myc-hPrp6 added to the reaction.
FIGURE 4.
FIGURE 4.
hPrp6 domains and protein–protein interactions. (A) Schematic representation of the protein fragments derived from full-length hPrp6 used in two-hybrid assays. (B) The yeast strain AH109 was transformed with pGBKT7 carrying hPrp6 fragments and pGADT7 carrying hDib1, hPrp31, hSnu66, and fragments hBrr2–4 and hPrp8–1. Empty vectors were included as a negative control. Combinations labeled ND were not analyzed due to self-activation of the bait. (Black squares) Interactions selected on SD/−His−Ade, (hatched squares) those selected on SD/−His. (C) Interactions of reciprocal bait–prey pairs. (D) Schematic drawing of the observed interactions involving hPrp6.
FIGURE 5.
FIGURE 5.
Analysis of interactions between the hSnu66 protein and in vitro-translated fusion proteins of (A) hPrp3 and hPrp6, and (B) full-length hBrr2, as well as fragments hBrr2–4, hBrr2–5. Purified His-tagged hSnu66 was incubated with anti-hSnu66 antibodies bound to protein A-Sepharose and [35S]methionine-labeled proteins produced by in vitro translation. The precipitated proteins were fractionated by SDS-PAGE and visualized by autoradiography. Mock precipitations without His-tagged hSnu66 protein were included as a negative control. (C) Summary of hSnu66 interactions.
FIGURE 6.
FIGURE 6.
Protein–protein interactions of the hPrp3 protein. (A) Schematic drawing of the protein fragments derived from the full-length hPrp3 protein. (B) Yeast two-hybrid analysis of interactions between hPrp3 bait constructs (labeled according to panel A) and hSnu66 prey. (C) In vitro pull-downs of hPrp3-derived constructs with GST-hPrp6 (performed as described in Fig. 3).
FIGURE 7.
FIGURE 7.
Schematic depiction of protein–protein interactions within the human tri-snRNP. All dots indicate protein–protein interactions demonstrated in this study. Those in black and white indicate interactions also observed between the respective orthologous proteins in yeast. The Snu114p-Prp8p interaction has been reported by Dix et al. (1998), Prp6p–Dib1p by Uetz et al. (2000), Prp3p–Prp4p by Ito et al. (2001), and Prp8p–Brr2p, Brr2p–Snu66p, Snu66p–Prp6p by van Nues and Beggs (2001).
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