The evolution of the Puf superfamily of proteins across the tree of eukaryotes

Abstract

Background: Eukaryotic gene expression is controlled by a number of RNA-binding proteins (RBP), such as the proteins from the Puf (Pumilio and FBF) superfamily (PufSF). These proteins bind to RNA via multiple Puf repeat domains, each of which specifically recognizes a single RNA base. Recently, three diversified PufSF proteins have been described in model organisms, each of which is responsible for the maturation of ribosomal RNA or the translational regulation of mRNAs; however, less is known about the role of these proteins across eukaryotic diversity. Results: Here, we investigated the distribution and function of PufSF RBPs in the tree of eukaryotes. We determined that the following PufSF proteins are universally conserved across eukaryotes and can be broadly classified into three groups: (i) Nop9 orthologues, which participate in the nucleolar processing of immature 18S rRNA; (ii) 'classical' Pufs, which control the translation of mRNA; and (iii) PUM3 orthologues, which are involved in the maturation of 7S rRNA. In nearly all eukaryotes, the rRNA maturation proteins, Nop9 and PUM3, are retained as a single copy, while mRNA effectors ('classical' Pufs) underwent multiple lineage-specific expansions. We propose that the variation in number of 'classical' Pufs relates to the size of the transcriptome and thus the potential mRNA targets. We further distinguished full set of PufSF proteins in divergent metamonad Giardia intestinalis and initiated their cellular and biochemical characterization. Conclusions: Our data suggest that the last eukaryotic common ancestor (LECA) already contained all three types of PufSF proteins and that 'classical' Pufs then underwent lineage-specific expansions.

Efficient CRISPR/Cas9-mediated gene disruption in the tetraploid protist Giardia intestinalis

“…Importantly, the latter also defined highly specific and efficient endogenous NLS that targets Cas9 from S. pyogenes into both G. intestinalis nuclei [ 23 ]. Specifically, to the periphery of the nucleus, which resembles the presumed position of the nucleolus [ 41 , 42 ]. Importantly, the stable expression of Cas9 occurs without apparent toxicity to cells, which has caused problems in other systems, including protist parasites, e.g.…”

The Role of Pumilio RNA Binding Protein in Plants

“…Pum classes were sorted by the numbers of the alpha helical repeats of the Pumilio-homology domain (Pum-HD) via SMART tool and were mainly represented as the canonical and non-canonical Pum-HD ( Figure 1 B) [ 21 ]. Pum-HD is necessary and sufficient for sequence-specific RNA binding [ 22 , 23 , 24 ]. Basically, the eight alpha helical repeats of Pum-HD could confer recognition and binding affinity to a Pumilio RNA binding motif of the target RNA [ 25 ].…”

Phosphorylation of PUF-A/PUM3 on Y259 modulates PUF-A stability and cell proliferation

“…HeLa cells were trypsinized and number counted by a hemocytometer. Totally, 1x10 3 control and PUF-A knockout HeLa cells were seeded in DMEM complete growth media and allowed to grow for 12 days until visible colonies formed. Colonies were stained with 0.25% crystal violet in ddH 2 O, washed with PBS twice, and air dried.…”

“…Pumilio/fem-3 (PUF) proteins belong to the members of Drosophila melanogaster Pumilio and Caenorhabditis elegans fem-3 mRNA binding factors [1,2], which contain of 8-12 conserved α-helical Pumilio (PUM) repeats [3,4]. Each PUM repeat consists of 35 to 39 amino acids capable of associating with the 3'-untranslated region (3'-UTR) of target mRNAs to promote mRNA degradation and translational repression [5][6][7][8][9].…”