Pseudouridine and N6-methyladenosine modifications weaken PUF protein/RNA interactions

When nutrients are depleted, Dictyostelium cells undergo cell cycle arrest and initiate a developmental program that ensures survival. The YakA protein kinase governs this transition by regulating the cell cycle, repressing growth-phase genes and inducing developmental genes. YakA mutants have a shortened cell cycle and do not initiate development. A suppressor of yakA that reverses most of the developmental defects of yakA- cells, but none of their growth defects was identified. The inactivated gene, pufA, encodes a member of the Puf protein family of translational regulators. Upon starvation, pufA- cells develop precociously and overexpress developmentally important proteins, including the catalytic subunit of cAMP-dependent protein kinase, PKA-C. Gel mobility-shift assays using a 200-base segment of PKA-C's mRNA as a probe reveals a complex with wild-type cell extracts, but not with pufA- cell extracts, suggesting the presence of a potential PufA recognition element in the PKA-C mRNA. PKA-C protein levels are low at the times of development when this complex is detectable, whereas when the complex is undetectable PKA-C levels are high. There is also an inverse relationship between PufA and PKA-C protein levels at all times of development in every mutant tested. Furthermore, expression of the putative PufA recognition elements in wild-type cells causes precocious aggregation and PKA-C overexpression, phenocopying a pufA mutation. Finally, YakA function is required for the decline of PufA protein and mRNA levels in the first 4 hours of development. We propose that PufA is a translational regulator that directly controls PKA-C synthesis and that YakA regulates the initiation of development by inhibiting the expression of PufA. Our work also suggests that Puf protein translational regulation evolved prior to the radiation of metazoan species.

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A Role for the Poly(A)-binding Protein Pab1p in PUF Protein-mediated Repression

Journal of Biological Chemistry ◽

10.1074/jbc.m111.264572 ◽

2011 ◽

Vol 286 (38) ◽

pp. 33268-33278 ◽

Cited By ~ 17

Author(s):

Jacqueline J. Chritton ◽

Marvin Wickens

Keyword(s):

Binding Protein ◽

Puf Protein

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Mpt5p, a Stress Tolerance- and Lifespan-Promoting PUF Protein in Saccharomyces cerevisiae, Acts Upstream of the Cell Wall Integrity Pathway

Eukaryotic Cell ◽

10.1128/ec.00188-06 ◽

2006 ◽

Vol 6 (2) ◽

pp. 262-270 ◽

Cited By ~ 30

Author(s):

Mark S. Stewart ◽

Sue Ann Krause ◽

Josephine McGhie ◽

Joseph V. Gray

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Wall ◽

Life Span ◽

Stress Tolerance ◽

Synthetic Medium ◽

Cell Wall Integrity ◽

Puf Proteins ◽

Downstream Target ◽

Cell Wall Integrity Pathway ◽

Puf Protein

ABSTRACT Pumilio family (PUF) proteins affect specific genes by binding to, and inhibiting the translation or stability of, their transcripts. The PUF domain is required and sufficient for this function. One Saccharomyces cerevisiae PUF protein, Mpt5p (also called Puf5p or Uth4p), promotes stress tolerance and replicative life span (the maximum number of doublings a mother cell can undergo before entering into senescence) by an unknown mechanism thought to partly overlap with, but to be independent of, the cell wall integrity (CWI) pathway. Here, we found that mpt5Δ mutants also display a short chronological life span (the time cells stay alive in saturated cultures in synthetic medium), a defect that is suppressed by activation of CWI signaling. We found that Mpt5p is an upstream activator of the CWI pathway: mpt5Δ mutants display the appropriate phenotypes and genetic interactions, display low basal activity of the pathway, and are defective in activation of the pathway upon thermal stress. A set of mRNAs that specifically bind to Mpt5p was recently reported. One such putative target, LRG1, encodes a GTPase-activating protein for Rho1p that directly links Mpt5p to CWI signaling: Lrg1p inhibits CWI signaling, LRG1 mRNA contains a consensus Mpt5p-binding site in its putative 3′ untranslated region, loss of Lrg1p suppresses the temperature sensitivity and CWI signaling defects of mpt5Δ mutants, and LRG1 mRNA abundance is inhibited by Mpt5p. We conclude that Mpt5p is required for normal replicative and chronological life spans and that the CWI pathway is a key and direct downstream target of this PUF protein.

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A Caenorhabditis elegans PUF protein family with distinct RNA binding specificity

RNA ◽

10.1261/rna.1095908 ◽

2008 ◽

Vol 14 (8) ◽

pp. 1550-1557 ◽

Cited By ~ 34

Author(s):

C. R. Stumpf ◽

J. Kimble ◽

M. Wickens

Keyword(s):

Caenorhabditis Elegans ◽

Rna Binding ◽

Binding Specificity ◽

Protein Family ◽

Puf Protein

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NcPuf1 Is a Key Virulence Factor in Neospora caninum

Pathogens ◽

10.3390/pathogens9121019 ◽

2020 ◽

Vol 9 (12) ◽

pp. 1019

Author(s):

Chenrong Wang ◽

Congshan Yang ◽

Jing Liu ◽

Qun Liu

Keyword(s):

Neospora Caninum ◽

Wide Distribution ◽

Key Factors ◽

Processing Bodies ◽

Puf Proteins ◽

P Bodies ◽

Intracellular Parasites ◽

Binding Factor ◽

Puf Protein

Background: Neospora caninum is an apicomplexan parasite that infects many mammals and particularly causes abortion in cattle. The key factors in its wide distribution are its virulence and ability to transform between tachyzoite and bradyzoite forms. However, the factors are not well understood. Although Puf protein (named after Pumilio from Drosophila melanogaster and fem-3 binding factor from Caenorhabditis elegans) have a functionally conserved role in promoting proliferation and inhibiting differentiation in many eukaryotes, the function of the Puf proteins in N. caninum is poorly understood. Methods: The CRISPR/CAS9 system was used to identify and study the function of the Puf protein in N. caninum. Results: We showed that N. caninum encodes a Puf protein, which was designated NcPuf1. NcPuf1 is found in the cytoplasm in intracellular parasites and in processing bodies (P-bodies), which are reported for the first time in N. caninum in extracellular parasites. NcPuf1 is not needed for the formation of P-bodies in N. caninum. The deletion of NcPuf1 (ΔNcPuf1) does not affect the differentiation in vitro and tissue cysts formation in the mouse brain. However, ΔNcPuf1 resulted in decreases in the proliferative capacity of N. caninum in vitro and virulence in mice. Conclusions: Altogether, the disruption of NcPuf1 does not affect bradyzoites differentiation, but seriously impairs tachyzoite proliferation in vitro and virulence in mice. These results can provide a theoretical basis for the development of attenuated vaccines to prevent the infection of N. caninum.

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