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. 2012 Mar 27;109(13):4846-50.
doi: 10.1073/pnas.1120792109. Epub 2012 Mar 13.

Structure of the Heterodimer of Human NONO and Paraspeckle Protein Component 1 and Analysis of Its Role in Subnuclear Body Formation

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Free PMC article

Structure of the Heterodimer of Human NONO and Paraspeckle Protein Component 1 and Analysis of Its Role in Subnuclear Body Formation

Daniel M Passon et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Proteins of the Drosophila behavior/human splicing (DBHS) family include mammalian SFPQ (PSF), NONO (p54nrb), PSPC1, and invertebrate NONA and Hrp65. DBHS proteins are predominately nuclear, and are involved in transcriptional and posttranscriptional gene regulatory functions as well as DNA repair. DBHS proteins influence a wide gamut of biological processes, including the regulation of circadian rhythm, carcinogenesis, and progression of cancer. Additionally, mammalian DBHS proteins associate with the architectural long noncoding RNA NEAT1 (Menε/β) to form paraspeckles, subnuclear bodies that alter gene expression via the nuclear retention of RNA. Here we describe the crystal structure of the heterodimer of the multidomain conserved region of the DBHS proteins, PSPC1 and NONO. These proteins form an extensively intertwined dimer, consistent with the observation that the different DBHS proteins are typically copurified from mammalian cells, and suggesting that they act as obligate heterodimers. The PSPC1/NONO heterodimer has a right-handed antiparallel coiled-coil that positions two of four RNA recognition motif domains in an unprecedented arrangement on either side of a 20-Å channel. This configuration is supported by a protein:protein interaction involving the NONA/paraspeckle domain, which is characteristic of the DBHS family. By examining various mutants and truncations in cell culture, we find that DBHS proteins require an additional antiparallel coiled-coil emanating from either end of the dimer for paraspeckle subnuclear body formation. These results suggest that paraspeckles may potentially form through self-association of DBHS dimers into higher-order structures.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Structure of a PSPC1/NONO heterodimer. (A) Domain structure indicating RNA recognition motifs 1 and 2, NOPS domain, and coiled-coil. (B) Stereoview of PSPC1 (cartoon) and NONO (surface). (C) Side (Left) and top (Right) views of PSPC1 (red) and NONO (green). (D) Cutaway view revealing voids (putative RNA-binding residues, cyan). (E) Side view highlighting putative RNA-binding residues of RRM1 (cyan).
Fig. 2.
Fig. 2.
Conserved residues in the NOPS domain (NONO: Y267, W271; PSPC1: Y275, W279) are critical for dimerization and paraspeckle localization. (A) The NOPS domain of PSPC1 (yellow) with Y275 and W279 circled. (B) Conserved Tyr and Trp residues have radically different side-chain conformations in NONO and PSPC1. (C) Wild-type Tyr and Trp, and not Ala, are required for paraspeckle formation by full-length YFP-NONO and mCherry-PSPC1 fusion proteins in HeLa cells. (Scale bars: 5 μm.) (D) Wild-type Tyr and Trp, and not Ala, are necessary for hetero- and homodimer formation in yeast two-hybrid analyses of full-length PSPC1/NONO (EYFP as negative control).
Fig. 3.
Fig. 3.
Coiled-coil formation is critical for paraspeckle targeting by DBHS proteins. (A) Observed coiled-coil (atomic model) and model of extended coiled-coil (cyan residues indicate hendecad repeat). (B) Sequence alignment of PSPC1, NONO, and SFPQ coiled coils; green arrows mark C termini of truncated PSPC1 proteins able to form paraspeckles; red arrows, those that cannot. (C) A schematic model for coiled-coil-mediated oligomerization of DBHS proteins. (D) YFP fusion proteins of PSPC1 proteins terminating after residue 337 colocalize with paraspeckles in HeLa cells; shorter proteins do not. (Scale bars: 5 μm.) (E) Wild-type endogenous NONO is coimmunoprecipitated by truncated YFP-PSPC1 proteins in an anti-GFP pull-down. Anti-GFP and anti-NONO Western blots.

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