Structural basis for snRNA recognition by the double-WD40 repeat domain of Gemin5

ABSTRACTHistone H3K4 methylation is an epigenetic mark associated with actively transcribed genes. This modification is catalyzed by the mixed lineage leukaemia (MLL) family of histone methyltransferases including MLL1, MLL2, MLL3, MLL4, SET1A and SET1B. Catalytic activity of MLL proteins is dependent on interactions with additional conserved proteins but the structural basis for subunit assembly and the mechanism of regulation is not well understood. We used a hybrid methods approach to study the assembly and biochemical function of the minimally active MLL1 complex (MLL1, WDR5 and RbBP5). A combination of small angle X-ray scattering (SAXS), cross-linking mass spectrometry (XL-MS), NMR spectroscopy, and computational modeling were used to generate a dynamic ensemble model in which subunits are assembled via multiple weak interaction sites. We identified a new interaction site between the MLL1 SET domain and the WD40 repeat domain of RbBP5, and demonstrate the susceptibility of the catalytic function of the complex to disruption of individual interaction sites.

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Basal body stability and ciliogenesis requires the conserved component Poc1

The Journal of Cell Biology ◽

10.1083/jcb.200908019 ◽

2009 ◽

Vol 187 (6) ◽

pp. 905-920 ◽

Cited By ~ 80

Author(s):

Chad G. Pearson ◽

Daniel P.S. Osborn ◽

Thomas H. Giddings ◽

Philip L. Beales ◽

Mark Winey

Keyword(s):

Cell Cycle ◽

Basal Body ◽

Tetrahymena Thermophila ◽

Wd40 Repeat ◽

Assembly Mechanism ◽

Repeat Domain ◽

Cilia Formation ◽

Centriole Assembly ◽

Protein Incorporation ◽

Dynamic Population

Centrioles are the foundation for centrosome and cilia formation. The biogenesis of centrioles is initiated by an assembly mechanism that first synthesizes the ninefold symmetrical cartwheel and subsequently leads to a stable cylindrical microtubule scaffold that is capable of withstanding microtubule-based forces generated by centrosomes and cilia. We report that the conserved WD40 repeat domain–containing cartwheel protein Poc1 is required for the structural maintenance of centrioles in Tetrahymena thermophila. Furthermore, human Poc1B is required for primary ciliogenesis, and in zebrafish, DrPoc1B knockdown causes ciliary defects and morphological phenotypes consistent with human ciliopathies. T. thermophila Poc1 exhibits a protein incorporation profile commonly associated with structural centriole components in which the majority of Poc1 is stably incorporated during new centriole assembly. A second dynamic population assembles throughout the cell cycle. Our experiments identify novel roles for Poc1 in centriole stability and ciliogenesis.

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Interactions of the dynein-2 intermediate chain WDR34 with the light chains are required for ciliary retrograde protein trafficking

Molecular Biology of the Cell ◽

10.1091/mbc.e18-10-0678 ◽

2019 ◽

Vol 30 (5) ◽

pp. 658-670 ◽

Cited By ~ 12

Author(s):

Yuta Tsurumi ◽

Yuki Hamada ◽

Yohei Katoh ◽

Kazuhisa Nakayama

Keyword(s):

Protein Trafficking ◽

Binding Sites ◽

Intraflagellar Transport ◽

Dominant Negative ◽

Light Chains ◽

Wd40 Repeat ◽

Phenotypic Differences ◽

Repeat Domain ◽

Ciliary Protein ◽

Intermediate Chain

The dynein-2 complex drives retrograde ciliary protein trafficking by associating with the intraflagellar transport (IFT) machinery, containing IFT-A and IFT-B complexes. We recently showed that the dynein-2 complex, which comprises 11 subunits, can be divided into three subcomplexes: DYNC2H1–DYNC2LI1, WDR34–DYNLL1/DYNLL2–DYNLRB1/DYNLRB2, and WDR60–TCTEX1D2–DYNLT1/DYNLT3. In this study, we demonstrated that the WDR34 intermediate chain interacts with the two light chains, DYNLL1/DYNLL2 and DYNLRB1/DYNLRB2, via its distinct sites. Phenotypic analyses of WDR34-knockout cells exogenously expressing various WDR34 constructs showed that the interactions of the WDR34 intermediate chain with the light chains are crucial for ciliary retrograde protein trafficking. Furthermore, we found that expression of the WDR34 N-terminal construct encompassing the light chain–binding sites but lacking the WD40 repeat domain inhibits ciliary biogenesis and retrograde trafficking in a dominant-negative manner, probably by sequestering WDR60 or the light chains. Taken together with phenotypic differences of several WDR34-knockout cell lines, these results indicate that incorporation of DYNLL1/DYNLL2 and DYNLRB1/DYNLRB2 into the dynein-2 complex via interactions with the WDR34 intermediate chain is crucial for dynein-2 function in retrograde ciliary protein trafficking.

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