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1.
bioRxiv ; 2024 Jun 14.
Article in English | MEDLINE | ID: mdl-38915480

ABSTRACT

PUF RNA-binding proteins are broadly conserved stem cell regulators. Nematode PUF proteins maintain germline stem cells (GSCs) and, with key partner proteins, repress differentiation mRNAs, including gld-1. Here we report that PUF protein FBF-2 and its partner LST-1 form a ternary complex that represses gld-1 via a pair of adjacent FBF-2 binding elements (FBEs) in its 3ÚTR. One LST-1 molecule links two FBF-2 molecules via motifs in the LST-1 intrinsically-disordered region; the gld-1 FBE pair includes a well-established 'canonical' FBE and a newly-identified noncanonical FBE. Remarkably, this FBE pair drives both full RNA repression in GSCs and full RNA activation upon differentiation. Discovery of the LST-1-FBF-2 ternary complex, the gld-1 adjacent FBEs, and their in vivo significance predicts an expanded regulatory repertoire of different assemblies of PUF-partner complexes in nematode germline stem cells. It also suggests analogous PUF controls may await discovery in other biological contexts and organisms.

2.
Mol Cell ; 82(21): 4049-4063.e6, 2022 11 03.
Article in English | MEDLINE | ID: mdl-36182693

ABSTRACT

In animals and plants, Dicer enzymes collaborate with double-stranded RNA-binding domain (dsRBD) proteins to convert precursor-microRNAs (pre-miRNAs) into miRNA duplexes. We report six cryo-EM structures of Drosophila Dicer-1 that show how Dicer-1 and its partner Loqs­PB cooperate (1) before binding pre-miRNA, (2) after binding and in a catalytically competent state, (3) after nicking one arm of the pre-miRNA, and (4) following complete dicing and initial product release. Our reconstructions suggest that pre-miRNA binds a rare, open conformation of the Dicer­1⋅Loqs­PB heterodimer. The Dicer-1 dsRBD and three Loqs­PB dsRBDs form a tight belt around the pre-miRNA, distorting the RNA helix to place the scissile phosphodiester bonds in the RNase III active sites. Pre-miRNA cleavage shifts the dsRBDs and partially closes Dicer-1, which may promote product release. Our data suggest a model for how the Dicer­1⋅Loqs­PB complex affects a complete cycle of pre-miRNA recognition, stepwise endonuclease cleavage, and product release.


Subject(s)
Drosophila Proteins , MicroRNAs , Animals , Ribonuclease III/genetics , Ribonuclease III/metabolism , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , RNA-Binding Proteins/metabolism , Drosophila/genetics , MicroRNAs/genetics , MicroRNAs/metabolism
3.
Nucleic Acids Res ; 47(16): 8770-8784, 2019 09 19.
Article in English | MEDLINE | ID: mdl-31294800

ABSTRACT

PUF proteins, named for Drosophila Pumilio (PUM) and Caenorhabditis elegans fem-3-binding factor (FBF), recognize specific sequences in the mRNAs they bind and control. RNA binding by classical PUF proteins is mediated by a characteristic PUM homology domain (PUM-HD). The Puf1 and Puf2 proteins possess a distinct architecture and comprise a highly conserved subfamily among fungal species. Puf1/Puf2 proteins contain two types of RNA-binding domain: a divergent PUM-HD and an RNA recognition motif (RRM). They recognize RNAs containing UAAU motifs, often in clusters. Here, we report a crystal structure of the PUM-HD of a fungal Puf1 in complex with a dual UAAU motif RNA. Each of the two UAAU tetranucleotides are bound by a Puf1 PUM-HD forming a 2:1 protein-to-RNA complex. We also determined crystal structures of the Puf1 RRM domain that identified a dimerization interface. The PUM-HD and RRM domains act in concert to determine RNA-binding specificity: the PUM-HD dictates binding to UAAU, and dimerization of the RRM domain favors binding to dual UAAU motifs rather than a single UAAU. Cooperative action of the RRM and PUM-HD identifies a new mechanism by which multiple RNA-binding modules in a single protein collaborate to create a unique RNA-binding specificity.


Subject(s)
RNA, Messenger/chemistry , RNA-Binding Proteins/chemistry , Schizosaccharomyces pombe Proteins/chemistry , Schizosaccharomyces/genetics , Amino Acid Sequence , Binding Sites , Cloning, Molecular , Crystallography, X-Ray , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Gene Library , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Models, Molecular , Nucleotide Motifs , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Protein Structure, Tertiary , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Schizosaccharomyces/metabolism , Schizosaccharomyces pombe Proteins/genetics , Schizosaccharomyces pombe Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino Acid
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