Post-transcriptional Regulatory Functions of Mammalian Pumilio Proteins

Abstract

Mammalian Pumilio proteins, PUM1 and PUM2, are members of the PUF family of sequence-specific RNA-binding proteins. In this review, we explore their mechanisms, regulatory networks, biological functions, and relevance to diseases. Pumilio proteins bind an extensive network of mRNAs and repress protein expression by inhibiting translation and promoting mRNA decay. Opposingly, in certain contexts, they can activate protein expression. Pumilio proteins also regulate noncoding (nc)RNAs. The ncRNA, ncRNA activated by DNA damage (NORAD), can in turn modulate Pumilio activity. Genetic analysis provides new insights into Pumilio protein function. They are essential for growth and development. They control diverse processes, including stem cell fate, and neurological functions, such as behavior and memory formation. Novel findings show that their dysfunction contributes to neurodegeneration, epilepsy, movement disorders, intellectual disability, infertility, and cancer.

Figure 1.. Features of classical cytoplasmic PUMs and divergent nucleolar PUMs.

(A) Diagrams of human PUM1 and PUM2 proteins showing length in amino acid (aa) residues, sequence motifs, secondary structure, disordered versus ordered regions (computed by JRONN) [119] and hydrophobic versus hydrophilic amino acid content (adapted from Protein Data Bank: https://www.rcsb.org). Post-translational modifications (PTM) including methylation, phosphorylation, and ubiquitylation from Uniprot (https://www.uniprot.org) and Phosphosite (https://www.phosphosite.org) are shown at the top. Motifs designated by a single letter represent low complexity regions enriched for that amino acid residue (A = alanine rich, Q = glutamine rich, S = serine rich, G = glycine rich). The TRMs within each Pum repeat (R1-R8) are also shown. (B) Diagram for founding member, Drosophila Pumilio, is shown for comparison. (C) Plot of relative sequence conservation versus amino acid residue position of 82 Pumilio protein orthologs including insects, fish, reptiles, birds, marsupials, mammals, primates and humans, generated using Clustal Omega [120], Consurf server [121], and Emboss Plotcon (http://www.bioinformatics.nl/cgi-bin/emboss/plotcon). For reference, conservation is plotted relative to functional domains defined for Drosophila Pumilio including three repression domains (RD1–3), Pumilio Conserved Motifs (PCMa and PCMb), and the Pum repeats (R1-R8) of the Pum- HD. Troughs represent sites of insertion. Peak height is proportional to conservation of sequence identity. (D) Diagrams of the divergent Pumilio orthologs PUM3 and NOP9. Motifs include predicted nuclear localization signals (NLS) and C-terminal Penguin Like (CPL) motif (PFam PF08144).

Figure 2.. Structure and RNA recognition by PUM1 and PUM2.

(A) High conservation of the RNA-binding surface of PUM proteins. Ribbon diagram of a crystal structure of human PUM1 in complex with hb NRE RNA is shown colored according to the degree of amino acid sequence conservation calculated using the Consurf server [117]. The most highly conserved positions are colored maroon, and the least conserved are colored cyan. The specific PUM1 amino acid residues that are mutated in PADDAS (R1139W and R1147W) and the PRCA (T1035S) are indicated by space-filling spheres. (B) PUM1 RNA recognition code. Specific interaction of Pum repeats with U, A, G, and C nucleotides is shown. (C) Ribbon diagram of a crystal structure of human PUM2 in complex with erk2 PRE RNA. The RNA-binding helices are colored maroon. PUM2 binds to the erk2 PRE RNA using the alternative ‘base-omission mode,’ where bases A4 and C5 (green) are directly stacked and R888 in repeat 5 (green) does not stack between bases A4 and C5.

Figure 3.. Model of PUM1/2 repression mechanisms.

PUM proteins bind the PRE, typically located in the 3´UTR of target mRNAs. Repression of the target mRNA is mediated in at least three ways: (A) The Pum-HDs of PUMs antagonize the translational activity of poly(A) binding protein (PABPC1). (B) PUMs promote translational repression and mRNA decay by recruiting the CCR4-NOT deadenylase complex (CNOT). (C) The N-terminal regions of PUM orthologs confer repression, but the mechanism remains to be determined.

Figure 4.. Gene expression of human PUM1 and PUM2 mRNAs across 53 tissue types.

Data were obtained from the GTEx consortium database. Violin plots showing median, interquartile range, and density for expression values of mRNAs for each PUM, in normalized, logio Transcripts per Million units (TPM), were generated for the indicated tissues using the GTEx portal website (http://www.gtexportal.org/) on 9/5/2018 using data from 10,294 samples.