PUF Family Of Proteins Research Articles

Principles of mRNA control by human PUM proteins elucidated from multimodal experiments and integrative data analysis.

The human PUF-family proteins, PUM1 and PUM2, posttranscriptionally regulate gene expression by binding to a PUM recognition element (PRE) in the 3′-UTR of target mRNAs. Hundreds of PUM1/2 targets have been identified from changes in steady-state RNA levels; however, prior studies could not differentiate between the contributions of changes in transcription and RNA decay rates. We applied metabolic labeling to measure changes in RNA turnover in response to depletion of PUM1/2, showing that human PUM proteins regulate expression almost exclusively by changing RNA stability. We also applied an in vitro selection workflow to precisely identify the binding preferences of PUM1 and PUM2. By integrating our results with prior knowledge, we developed a “rulebook” of key contextual features that differentiate functional versus nonfunctional PREs, allowing us to train machine learning models that accurately predict the functional regulation of RNA targets by the human PUM proteins.

Functions, mechanisms and regulation of Pumilio/Puf family RNA binding proteins: a comprehensive review.

The Pumilio (Pum)/Puf family proteins are ubiquitously present across eukaryotes, including yeast, plants and humans. They generally bind to the 3' untranslated regions of single stranded RNA targets in a sequence specific manner and destabilize them, although a few reports suggest their role in stabilizing the target transcripts. The Pum isoforms are implicated in a wide array of biological processes including stem cell maintenance, development, ribosome biogenesis as well as human diseases. Further studies on Pum would be interesting and important to understand their evolutionarily conserved and divergent features across species, which can have potential implications in medicine, plant sciences as well as basic molecular and cell biological studies. A large number of research reports exists, pertaining to various aspects of Pum, in individual experimental systems. This review is a comprehensive summary of the functions, types, mechanism of action as well as the regulation of Pum in various species. Also, the research questions to be addressed in future are discussed.

The target specificity of the RNA binding protein Pumilio is determined by distinct co-factors.

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 collaboration with cofactors.

Demonstration of protein cooperativity mediated by RNA structure using the human protein PUM2

Posttranslational gene regulation requires a complex network of RNA–protein interactions. Cooperativity, which tunes response sensitivities, originates from protein–protein interactions in many systems. For RNA-binding proteins, cooperativity can also be mediated through RNA structure. RNA structural cooperativity (RSC) arises when binding of one protein induces a redistribution of RNA conformational states that enhance access (positive cooperativity) or block access (negative cooperativity) to additional binding sites. As RSC does not require direct protein–protein interactions, it allows cooperativity to be tuned for individual RNAs, via alterations in sequence that alter structural stability. Given the potential importance of this mechanism of control and our desire to quantitatively dissect features that underlie physiological regulation, we developed a statistical mechanical framework for RSC and tested this model by performing equilibrium binding measurements of the human PUF family protein PUM2. Using 68 RNAs that contain two to five PUM2-binding sites and RNA structures of varying stabilities, we observed a range of structure-dependent cooperative behaviors. To test our ability to account for this cooperativity with known physical constants, we used PUM2 affinity and nearest-neighbor RNA secondary structure predictions. Our model gave qualitative agreement for our disparate set of 68 RNAs across two temperatures, but quantitative deviations arise from overestimation of RNA structural stability. Our results demonstrate cooperativity mediated by RNA structure and underscore the power of quantitative stepwise experimental evaluation of mechanisms and computational tools.

PUF-8 Functions Redundantly with GLD-1 to Promote the Meiotic Progression of Spermatocytes in Caenorhabditis elegans.

Successful meiotic progression of germ cells is crucial for gametogenesis. Defects in this process affect proper genetic transmission and sometimes lead to tumor formation in the germline. In Caenorhabditis elegans, the RNA-binding protein GLD-1 is essential for the meiotic development of oocytes. However, its role during spermatogenesis has not been understood. Here, we show that GLD-1 functions redundantly with the PUF family protein PUF-8 to ensure proper meiotic development of spermatocytes. When grown at 20°—the standard laboratory temperature for C. elegans growth—primary spermatocytes in both gld-1 and puf-8 single-mutant males and hermaphrodites complete the meiotic divisions normally. By contrast, some of the gld-1; puf-8 double-mutant spermatocytes exit meiosis and form germ cell tumors in both sexes. During larval development, gld-1; puf-8 double-mutant germ cells begin to express the meiotic marker HIM-3, lose P granules, and form the sperm-specific membranous organelle, which are characteristics of developing spermatocytes. However, some of these cells quickly lose HIM-3 and form germ cell tumors that lack membranous organelle but contain P granules. Mutations that block meiotic progression at late pachytene or diakinetic stage fail to arrest the tumorigenesis, suggesting that the gld-1; puf-8 double-mutant spermatocytes exit meiosis prior to the completion of pachytene. Together, results presented here uncover a novel function for gld-1 in the meiotic development of spermatocytes in both hermaphrodites and males.

APUM23, a PUF family protein, functions in leaf development and organ polarity in Arabidopsis

The normal biological function of leaves, such as intercepting light and exchanging gases, relies on proper differentiation of adaxial and abaxial polarity. KANADI (KAN) genes, members of the GARP family, are key regulators of abaxial identity in leaf morphogenesis. This study identified a mutant allele (apum23-3) of APUM23, which encodes a Pumilio/PUF domain protein and acts as an enhancer of the kan mutant. Arabidopsis APUM23 has been shown to function in pre-rRNA processing and play pleiotropic roles in plant development. The apum23-3 mutant also synergistically interacts with other leaf polarity mutants, affects proliferation of division-competent cells, and alters the expression of important leaf polarity genes. These phenotypes show that APUM23 has critical functions in plant development, particularly in polarity formation. The PUF gene family is conserved across kingdoms yet it has not been well characterized in plants. These results illuminating the functions of APUM23 suggest a novel role for PUF genes in Arabidopsis leaf development.

PUF-8 and TCER-1 are essential for normal levels of multiple mRNAs in the C. elegans germline.

PUF family proteins are well-conserved regulators of cell proliferation in different developmental processes. They regulate target mRNAs by promoting degradation or by influencing translation through interaction with the translation initiation machinery. Here we show that Caenorhabditis elegans PUF-8 functions redundantly with the nuclear protein TCER-1 in the post-transcriptional maintenance of at least six germline mRNAs. The levels of spliced mRNAs in the puf-8(-) tcer-1(-) double mutant are only 10-30% of the wild type, whereas the unspliced forms increase by ∼2- to 3-fold compared with the wild type. These two proteins colocalise at the inner nuclear periphery, and their absence leads to reduced germ cell proliferation and to sterility. A yeast two-hybrid screen of 31 components of the nuclear pore complex and mRNA processing machineries identified seven proteins involved in mRNA export as potential partners of PUF-8. One of these, the nuclear cap-binding protein NCBP-2, colocalises with PUF-8 in the nucleus. A 50 amino acid N-terminal domain of PUF-8 is essential for interaction with NCBP-2 and for PUF-8 to function redundantly with TCER-1. These results reveal two important unexpected aspects of PUF proteins: that, in addition to the C-terminal PUF domain, the N-terminal domain is crucial for PUF function, and that PUF proteins have a novel role in mRNA maintenance. We propose that PUF proteins, in addition to their known cytoplasmic roles, participate in nuclear processing and/or export of mRNAs.

The Puf-family RNA-binding protein PfPuf2 regulates sexual development and sex differentiation in the malaria parasitePlasmodium falciparum

Translation regulation plays an important role during gametocytogenesis in the malaria parasite, a process that is obligatory for the transmission of the parasite through mosquito vectors. In this study we determined the function of PfPuf2, a member of the Puf family of translational repressors, in gametocytogenesis of Plasmodium falciparum. Tagging of the endogenous PfPuf2 protein with green fluorescent protein showed that PfPuf2 was expressed in both male and female gametocytes, and the protein was localized in the cytoplasm of the parasite. Targeted disruption of the PfPuf2 gene did not affect asexual growth of the parasite, but promoted the formation of gametocytes and differentiation of male gametocytes. Complementation studies were performed to confirm that the resultant phenotypic changes were due to disruption of the PfPuf2 gene. Episomal expression of PfPuf2 under its cognate promoter almost restored the gametocytogenesis rate in a PfPuf2 disruptant to the level of the wild-type parasite. It also partially restored the effect of PfPuf2 disruption on male-female sex ratio. In addition, episomal overexpression of PfPuf2 under its cognate promoter but with a higher concentration of the selection drug or under the constitutive hsp86 promoter in both the PfPuf2-disruptant and wild-type 3D7 lines, further dramatically reduced gametocytogenesis rates and sex ratios. These findings suggest that in this early branch of eukaryotes the function of PfPuf2 is consistent with the ancestral function of suppressing differentiation proposed for Puf-family proteins.

Comparative Analysis of mRNA Targets for Human PUF-Family Proteins Suggests Extensive Interaction with the miRNA Regulatory System

Genome-wide identification of mRNAs regulated by RNA-binding proteins is crucial to uncover post-transcriptional gene regulatory systems. The conserved PUF family RNA-binding proteins repress gene expression post-transcriptionally by binding to sequence elements in 3′-UTRs of mRNAs. Despite their well-studied implications for development and neurogenesis in metazoa, the mammalian PUF family members are only poorly characterized and mRNA targets are largely unknown. We have systematically identified the mRNAs associated with the two human PUF proteins, PUM1 and PUM2, by the recovery of endogenously formed ribonucleoprotein complexes and the analysis of associated RNAs with DNA microarrays. A largely overlapping set comprised of hundreds of mRNAs were reproducibly associated with the paralogous PUM proteins, many of them encoding functionally related proteins. A characteristic PUF-binding motif was highly enriched among PUM bound messages and validated with RNA pull-down experiments. Moreover, PUF motifs as well as surrounding sequences exhibit higher conservation in PUM bound messages as opposed to transcripts that were not found to be associated, suggesting that PUM function may be modulated by other factors that bind conserved elements. Strikingly, we found that PUF motifs are enriched around predicted miRNA binding sites and that high-confidence miRNA binding sites are significantly enriched in the 3′-UTRs of experimentally determined PUM1 and PUM2 targets, strongly suggesting an interaction of human PUM proteins with the miRNA regulatory system. Our work suggests extensive connections between the RBP and miRNA post-transcriptional regulatory systems and provides a framework for deciphering the molecular mechanism by which PUF proteins regulate their target mRNAs.

The RNA-binding proteins PUF-5, PUF-6, and PUF-7 reveal multiple systems for maternal mRNA regulation during C. elegans oogenesis

In metazoans, many mRNAs needed for embryogenesis are produced during oogenesis and must be tightly regulated during the complex events of oocyte development. In C. elegans, translation of the Notch receptor GLP-1 is repressed during oogenesis and is then activated specifically in anterior cells of the early embryo. The KH domain protein GLD-1 represses glp-1 translation during early stages of meiosis, but the factors that repress glp-1 during late oogenesis are not known. Here, we provide evidence that the PUF domain protein PUF-5 and two nearly identical PUF proteins PUF-6 and PUF-7 function during a specific period of oocyte differentiation to repress glp-1 and other maternal mRNAs. Depletion of PUF-5 and PUF-6/7 together caused defects in oocyte formation and early embryonic cell divisions. Loss of PUF-5 and PUF-6/7 also caused inappropriate expression of GLP-1 protein in oocytes, but GLP-1 remained repressed in meiotic germ cells. PUF-5 and PUF-6/7 function was required directly or indirectly for translational repression through elements of the glp-1 3′ untranslated region. Oogenesis and embryonic defects could not be rescued by loss of GLP-1 activity, suggesting that PUF-5 and PUF-6/7 regulate other mRNAs in addition to glp-1. PUF-5 and PUF-6/7 depletion, however, did not perturb repression of the maternal factors GLD-1 and POS-1, suggesting that subsets of maternal gene products may be regulated by distinct pathways. Interestingly, PUF-5 protein was detected exclusively during mid to late oogenesis but became undetectable prior to completion of oocyte differentiation. These results reveal a previously unknown maternal mRNA control system that is specific to late stages of oogenesis and suggest new functions for PUF family proteins in post-mitotic differentiation. Multiple sets of RNA-binding complexes function in different domains of the C. elegans germ line to maintain silencing of Notch/glp-1 and other mRNAs.

Suppressor analysis of the mpt5/htr1/uth4/puf5 deletion in Saccharomyces cerevisiae

The MPT5/HTR1/UTH4/PUF5 gene encodes an RNA-binding Puf-family protein in Saccharomyces cerevisiae. The Deltampt5 cells exhibit pleiotropic phenotypes, including the G2/M arrest of the cell cycle and weakened cell wall at high temperatures. The Deltampt5 disruptant was also hydroxyurea (HU) sensitive. In this study we screened deletion suppressors to rescue the temperature sensitivity of Deltampt5, and identified dsf1 (YEL070W), dsf2 (YBR007C), sir2, sir3, sir4 and swe1. Multicopy suppressors identified were PKC1 and its upstream genes, but not the downstream MAPK cascade genes. The overexpression of PKC1, however, did not suppress the HU sensitivity of Deltampt5. In contrast, both the HU- and temperature-sensitivities of a-type Deltampt5 cells were suppressed by each sir deletion or a multicopy of MATalpha2, suggesting that a diploid-type expression is involved. We found that a diploid-specific IME4 gene encoding an RNA-modifying protein was responsible for the suppression of the temperature sensitivity, but not of the HU sensitivity. Furthermore, the suppression of the HU sensitivity depended on PUF4, another Puf-family gene, and overexpression of PUF4 suppressed only the HU sensitivity of Deltampt5. The protein level of Puf4 was not affected by the sir mutation. Thus, these Ime4 and Puf4 proteins play complementary roles to rescue the defects in Deltampt5 Deltasir cells.

Characterization of PfPuf2, Member of the Puf Family RNA-Binding Proteins from the Malaria ParasitePlasmodium falciparum

Puf proteins are a family of evolutionarily conserved translational regulators in eukaryotes. The malaria parasite has two Puf proteins (PfPuf1 and PfPuf2) that share 25% homology in the RNA binding domain. Here we confirmed the preferential expression of PfPuf2 in gametocyte stages using Northern analysis. The transcriptional initiation site of this gene, mapped using RNA ligase-mediated rapid amplification of cDNA end and primer extension, is located approximately 300 bp upstream from the translational start codon. The 3' end of PfPuf2 is located approximately 250 bp downstream from the stop codon. The total length of the RNA is approximately 2.1 kb, consistent with the mRNA size determined by Northern analysis. Recombinant PfPuf2 proteins expressed in bacteria were purified and used to produce polyclonal antibodies. Western blot further established the preferential synthesis of PfPuf2 in gametocyte stages. Using the Nanos-responsive elements (NRE) in the Hunchback mRNA of Drosophila melanogaster as an artificial target sequence, we tested the binding of PfPuf2 Puf domain to this sequence using the yeast three-hybrid system. The results showed that PfPuf2 Puf domain bound specifically to NRE, suggesting that PfPuf2 may be involved in translational regulation of target genes using a conserved mechanism of the Puf family proteins.

A new yeast PUF family protein, Puf6p, represses ASH1 mRNA translation and is required for its localization.

In yeast Saccharomyces cerevisiae, Ash1p, a protein determinant for mating-type switching, is segregated within the daughter cell nucleus to establish asymmetry of HO expression. The accumulation of Ash1p results from ASH1 mRNA that is sorted as a ribonucleoprotein particle (mRNP or locasome) to the distal tip of the bud where translation occurs. To study the mechanism regulating ASH1 mRNA translation, we isolated the ASH1 locasome and characterized the associated proteins by MALDI-TOF. One of these proteins was Puf6p, a new member of the PUF family of highly conserved RNA-binding proteins such as Pumilio in Drosophila, responsible for translational repression, usually to effect asymmetric expression. Puf6p-bound PUF consensus sequences in the 3'UTR of ASH1 mRNA and repressed the translation of ASH1 mRNA both in vivo and in vitro. In the puf6 Delta strain, asymmetric localization of both Ash1p and ASH1 mRNA were significantly reduced. We propose that Puf6p is a protein that functions in the translational control of ASH1 mRNA, and this translational inhibition is necessary before localization can proceed.

Trypanosoma brucei expression-site-associated-gene-8 protein interacts with a Pumilio family protein

The expression site (ES) loci of Trypanosoma brucei are a valuable model for allelic exclusion and post-transcriptional regulation in a highly divergent eukaryote. ES exist to facilitate the expression and switching of the variant surface glycoproteins (VSG) that are central to trypanosome virulence and persistence. A collection of other potential virulence determinants, known as expression-site-associated-genes (ESAGs), are co-transcribed from the single upstream promoter. ESAGs may be involved in regulating the transcriptional state of the ES, as well as contributing additional surface proteins and receptors. We have previously shown that a putative regulatory protein, ESAG8, accumulates within the nucleolus, although 20% of the protein is cytoplasmic. Here we identify TbPUF1, a cytoplasmic ESAG8-interacting protein that falls into the Puf family of regulators of mRNA stability. Our experiments show that, as in other Puf family proteins, the most C-terminal repeats of TbPUF1 mediate its interaction with ESAG8. TbPUF1 is essential for cell viability, and preliminary results suggest that its overexpression seriously affects parasite virulence. T. brucei is the most evolutionary divergent organism in which a Puf family protein has been identified, and our initial experiments suggest that this protein may also regulate RNA stability in trypanosomes.