A divergent Pumilio repeat protein family for pre-rRNA processing and mRNA localization (original) (raw)
Related papers
IUBMB Life (International Union of Biochemistry and Molecular Biology: Life), 2003
Drosophila Pumilio (Pum) protein is a founder member of a novel family of RNA-binding proteins, known as the PUF family. The PUF proteins constitute an evolutionarily highly conserved family of proteins present from yeast to humans and plants, and are characterized by a highly conserved C-terminal RNA-binding domain, composed of eight tandem repeats. The conserved biochemical features and genetic function of PUF family members have emerged from studies of model organisms. PUF proteins bind to related sequence motifs in the 3' untranslated region (3'UTR) of specific target mRNAs and repress their translation. Frequently, PUF proteins function asymmetrically to create protein gradients, thus causing asymmetric cell division and regulating cell fate specification. Thus, it was recently proposed that the primordial role of PUF proteins is to sustain mitotic proliferation of stem cells. Here we review the evolution, conserved genetic and biochemical properties of PUF family of proteins, and discuss protein interactions, upstream regulators and downstream targets of PUF proteins. We also suggest that a conserved mechanism of PUF function extends to the newly described mammalian members of the PUF family (human PUM1 and PUM2, and mouse Pum1 and Pum2), that show extensive homology to Drosophila Pum, and could have an important role in cell development, fate specification and differentiation.
Journal of Biological Chemistry, 2011
Background: PUF protein RNA recognition is critical for target gene regulation. Results: A chemically conserved binding pocket in a subset of PUF proteins recognizes cytosine at different positions upstream of the core PUF recognition sequence. Conclusion: A specialized cytosine-binding pocket introduces qualitative and quantitative differences in RNA recognition by PUF proteins. Significance: Simple adaptations can diversify PUF protein RNA recognition.
Specific and modular binding code for cytosine recognition in Pumilio/FBF (PUF) RNA-binding domains
2011
Pumilio/fem-3 mRNA-binding factor (PUF) proteins possess a recognition code for bases A, U, and G, allowing designed RNA sequence specificity of their modular Pumilio (PUM) repeats. However, recognition side chains in a PUM repeat for cytosine are unknown. Here we report identification of a cytosine-recognition code by screening random amino acid combinations at conserved RNA recognition positions using a yeast three-hybrid system. This C-recognition code is specific and modular as specificity can be transferred to different positions in the RNA recognition sequence. A crystal structure of a modified PUF domain reveals specific contacts between an arginine side chain and the cytosine base. We applied the C-recognition code to design PUF domains that recognize targets with multiple cytosines and to generate engineered splicing factors that modulate alternative splicing. Finally, we identified a divergent yeast PUF protein, Nop9p, that may recognize natural target RNAs with cytosine. This work deepens our understanding of natural PUF protein target recognition and expands the ability to engineer PUF domains to recognize any RNA sequence.
Gene, 2002
Drosophila gene Pumilio (Pum) is a founder member of an evolutionarily conserved family of RNA-binding proteins that are present from yeast to mammals, and act as translational repressors during embryo development and cell differentiation. The human genome contains two Pumilio related genes, PUM1 and PUM2, that encode 127 and 114 kDa proteins with evolutionarily highly conserved Pum RNA-binding domain (86 and 88% homology with the fly Pum protein). PUM1 and PUM2 proteins share 83% overall similarity, with RNA-binding domain being 91% identical. Both PUM1 and PUM2 show relatively widespread and mostly overlapping expression in human tissues, and are very large genes with highly conserved gene structure. PUM1 consists of 22 exons, spanning about 150 kb on chromosome 1p35.2, whereas PUM2 consists of 20 exons and spans at least 80 kb on chromosome 2p23-24. Extremely high evolutionary conservation of the RNA-binding domain from yeast to humans, and conserved function of Pumilio proteins in invertebrates and lower vertebrates suggest that mammalian Pumilio proteins could also play an important role in translational regulation of embryogenesis and cell development and differentiation.
Crystallization and Characterization of Pumilio: A Novel RNA Binding Protein
Journal of Structural Biology, 2000
Drosophila embryos is controlled, in part, by regulation of translation of mRNAs transcribed in maternal cells during oogenesis. The Pumilio protein is essential in posterior determination, binding to hunchback mRNA in complex with Nanos to suppress hunchback translation. In order to understand the structural basis of RNA binding, Nanos recruitment, and translational control, we have crystallized a domain of the Drosophila Pumilio protein that binds RNA. The crystals belong to the space group P6 3 with unit cell dimensions of a ؍ b ؍ 94.5 Å, c ؍ 228.9 Å, ␣ ؍  ؍ 90°, ␥ ؍ 120°and diffract to 2.6 Å with synchrotron radiation. We show that the purified protein actively binds RNA and is likely to have a novel RNA binding fold due to a very high content of ␣-helical secondary structure.
Crystallization and characterization of Pumilo: a novel RNA binding protein
Journal of structural biology, 2000
Drosophila embryos is controlled, in part, by regulation of translation of mRNAs transcribed in maternal cells during oogenesis. The Pumilio protein is essential in posterior determination, binding to hunchback mRNA in complex with Nanos to suppress hunchback translation. In order to understand the structural basis of RNA binding, Nanos recruitment, and translational control, we have crystallized a domain of the Drosophila Pumilio protein that binds RNA. The crystals belong to the space group P6 3 with unit cell dimensions of a ؍ b ؍ 94.5 Å, c ؍ 228.9 Å, ␣ ؍  ؍ 90°, ␥ ؍ 120°and diffract to 2.6 Å with synchrotron radiation. We show that the purified protein actively binds RNA and is likely to have a novel RNA binding fold due to a very high content of ␣-helical secondary structure.
Alternative Conformations at the RNA-binding Surface of the N-terminal U2AF65 RNA Recognition Motif
Journal of Molecular Biology, 2007
The essential pre-mRNA splicing factor, U2 auxiliary factor 65KD (U2AF 65 ) recognizes the polypyrimidine tract (Py-tract) consensus sequence of the pre-mRNA using two RNA recognition motifs (RRMs), the most prevalent class of eukaryotic RNA-binding domain. The Py-tracts of higher eukaryotic pre-mRNAs are often interrupted with purines, yet U2AF 65 must identify these degenerate Py-tracts for accurate pre-mRNA splicing. Previously, the structure of a U2AF 65 variant in complex with poly(U) RNA suggested that rearrangement of flexible side-chains or bound water molecules may contribute to degenerate Py-tract recognition by U2AF 65 . Here, the X-ray structure of the N-terminal RRM domain of U2AF 65 (RRM1) is described at 1.47 Å resolution in the absence of RNA. Notably, RNA-binding by U2AF 65 selectively stabilizes pre-existing alternative conformations of three sidechains located at the RNA interface (Arg150, Lys225, and Arg227). Additionally, a flexible loop connecting the β2/β3 strands undergoes a conformational change to interact with the RNA. These pre-existing alternative conformations may contribute to the ability of U2AF 65 to recognize a variety of Py-tract sequences. This rare, high-resolution view of an important member of the RRM class of RNA-binding domains highlights the role of alternative side-chain conformations in RNA recognition.
The target specificity of the RNA binding protein Pumilio is determined by distinct co-factors
Bioscience Reports, 2019
Puf family proteins are translational regulators essential to a wide range of biological processes, including cell fate specification, stem cell self-renewal, and neural function. Yet, despite being associated with hundreds of RNAs, the underlying mechanisms of Puf target specification remain to be fully elucidated. In Drosophila, Pumilio – a sole Puf family protein – is known to collaborate with cofactors Nanos (Nos) and Brain Tumor (Brat); however, their roles in target specification are not clearly defined. Here, we identify Bag-of-marbles (Bam) as a new Pum cofactor in repression of Mothers against dpp (mad) mRNAs, for which Nos is known to be dispensable. Notably, our data show that Nos (but not Bam) was required for Pum association with hunchback (hb) mRNAs, a well-known target of Pum and Nos. In contrast, Bam (but not Nos) was required for Pum association with mad mRNAs. These findings show for the first time that Pum target specificity is determined not independently but in ...
FEBS Journal, 2005
The RNA recognition motif (RRM), also known as the RNA-binding domain (RBD) or ribonucleoprotein domain (RNP), was first identified in the late 1980s when it was demonstrated that mRNA precursors (pre-mRNA) and heterogeneous nuclear RNAs (hnRNAs) are always found in complex with proteins (reviewed in [1]). Biochemical characterizations of the mRNA polyadenylate binding protein (PABP) and the hnRNP protein C shed light on a consensus RNA-binding domain of approximately 90 amino acids containing a central sequence of eight conserved residues that are mainly aromatic and positively charged . This sequence, termed the RNP consensus sequence, was thought to be involved in RNA interaction and was defined as Lys ⁄ Arg-Gly-Phe ⁄ Tyr-Gly ⁄ Ala-Phe ⁄ Tyr-Val ⁄ Ile ⁄ Leu-X-Phe ⁄ Tyr, where X can be any amino acid. Later, a second consensus sequence less conserved than the previously characterized one [1] was identified. This six residue sequence located at the N-terminus of the domain The RNA recognition motif (RRM), also known as RNA-binding domain (RBD) or ribonucleoprotein domain (RNP) is one of the most abundant protein domains in eukaryotes. Based on the comparison of more than 40 structures including 15 complexes (RRM-RNA or RRM-protein), we reviewed the structure-function relationships of this domain. We identified and classified the different structural elements of the RRM that are important for binding a multitude of RNA sequences and proteins. Common structural aspects were extracted that allowed us to define a structural leitmotif of the RRM-nucleic acid interface with its variations. Outside of the two conserved RNP motifs that lie in the center of the RRM b-sheet, the two external b-strands, the loops, the C-and N-termini, or even a second RRM domain allow high RNA-binding affinity and specific recognition. Protein-RRM interactions that have been found in several structures reinforce the notion of an extreme structural versatility of this domain supporting the numerous biological functions of the RRM-containing proteins.