The catalytic-dead Pcif1 regulates gene expression and fertility in Drosophila

Abstract

Eukaryotic mRNAs are modified at the 5' end with a methylated guanosine (m⁷G) that is attached to the transcription start site (TSS) nucleotide. The TSS nucleotide is 2'-O-methylated (Nm) by CMTR1 in organisms ranging from insects to human. In mammals, the TSS adenosine can be further N ⁶ -methylated by RNA polymerase II phosphorylated CTD-interacting factor 1 (PCIF1) to create m⁶Am. Curiously, the fly ortholog of mammalian PCIF1 is demonstrated to be catalytic-dead, and its functions are not known. Here, we show that Pcif1 mutant flies display a reduced fertility which is particularly marked in females. Deep sequencing analysis of Pcif1 mutant ovaries revealed transcriptome changes with a notable increase in expression of genes belonging to the mitochondrial ATP synthetase complex. Furthermore, the Pcif1 protein is distributed along euchromatic regions of polytene chromosomes, and the Pcif1 mutation behaved as a modifier of position-effect-variegation (PEV) suppressing the heterochromatin-dependent silencing of the white gene. Similar or stronger changes in the transcriptome and PEV phenotype were observed in flies that expressed a cytosolic version of Pcif1. These results point to a nuclear cotranscriptional gene regulatory role for the catalytic-dead fly Pcif1 that is probably based on its conserved ability to interact with the RNA polymerase II carboxy-terminal domain.

Keywords: N6-methyladenosine; Pcif1; m6A; m6Am; position-effect variegation (PEV); transcription.

FIGURE 1.

Molecular characterization of Pcif1 in Drosophila melanogaster. (A) Structural organization of the wild-type Pcif1 protein. The amino-terminal WW domain, the nuclear localization signal (NLS), the helical domain, and the carboxy-terminal methyl-transferase (MTase) domain are shown. (B) The CRISPR–Cas9 mutagenesis generated the Pcif1^M6stop allele, which causes a frameshift ending in a premature stop codon generating a truncated protein and the Pcif1^F9ΔNLS allele which has an in-frame deletion of the NLS. (C) Illustration of the Pcif1 locus with the indication of the P element insertion site in the Pcif1^BG02557 line (Dmel\PCG11399^BG02557), here used as control, and of the position of the sgRNAs, gRNA1, and gRNA2 designed for the CRISPR–Cas9 mutagenesis (purple lines). (D) Western blot analysis of head protein extracts from Pcif1^BG02557 control, or Pcif1^M6stop, and Pcif1^F9ΔNLS mutants as indicated. Wild-type Pcif1 and Pcif1^ΔNLS protein sizes are indicated by the black arrows. β-Actin was used as loading control. The asterisks indicate unspecific bands. (E) Immunostaining of ovaries dissected from Pcif1^BG02557 controls, Pcif1^F9ΔNLS homozygous, or Pcif1^F9ΔNLS/+ heterozygous females. The egg chamber, formed by an external layer of follicle cells (somatic cells) that surround the nurse cells (germ cells), is shown. Pcif1: green, DNA: blue, Fibrillarin (nucleolar marker): red. (F) Pictures of 5-d-old females, males, or dissected ovaries of either Pcif1^BG02557 (control) or Pcif1^M6stop and Pcif1^F9ΔNLS mutants. (G) Longevity analysis of Pcif1^BG02557 control (n = 150) or Pcif1^M6stop (n = 150) and Pcif1^F9ΔNLS (n = 130) mutants. (H) Weight analysis of Pcif1^BG02557 control or Pcif1^M6stop and Pcif1^F9ΔNLS mutants: each dot represents the weight of a group of 10 adult flies per genotype for either males or females as indicated. Ten replicates were performed. (I) Fertility analysis of females or males expressed as the number of emerging flies per cross of one individual of the indicated genotype with three wild-type flies of the opposite sex. Log-rank (longevity) and one-way ANOVA (weight and fertility) tests were performed using GraphPad Prism9. P-value: (***) P < 0.001, (****) P < 0.0001. ns: nonsignificant.

FIGURE 2.

Transcriptomic analyses in Pcif1 mutants. (A) Barplot showing reduced transcript levels of Pcif1 in Pcif1^M6stop while the transcript levels in Pcif1^F9ΔNLS are not affected. Data shown as mean ± standard deviation from three biological replicas. (B) MA plots and volcano plots showing gene expression changes in Pcif1^M6stop and in Pcif1^F9ΔNLS. Significantly dysregulated genes (P_adj ≤ 0.1) are shown in red. (C) Boxplots showing gene expression changes from individual chromosomes. The view is limited to only changes below twofold to better see the median differences (P-value: [*] P < 0.05, [***] P < 0.001; wilcox.text of symmetric distribution). (D) Venn diagrams showing overlap in sets of up-regulated and down-regulated genes in Pcif1^M6stop and Pcif1^F9ΔNLS.

FIGURE 3.

Pcif1 regulates gene expression of proton transport genes. (A) Gene ontology analysis for differentially expressed genes. Only the top 10 significantly enriched terms are shown. (B) Boxplot showing the z-scores of expression across individual samples for proton transport genes (GO:0015992). (C) Heatmap showing the z-scores of expression for proton transport genes (GO:0015992). (D) Plots showing the cumulative distributions of gene expression changes in the Pcif1^M6stop and in Pcif1^F9ΔNLS when compared to the control. Separate curves are plotted based on the transcription start site nucleotide (TSS).

FIGURE 4.

Pcif1 binds to euchromatic regions of polytene chromosomes and behaves as a suppressor of position-effect variegation. (A) Larval polytene chromosome immunostaining was performed for Pcif1^BG02557 controls (two samples) and Pcif1^M6stop mutants as indicated. (B) PEV assay. Schematic illustration of the position-effect variegation affecting the white gene in the w^m4h allele. Representative eye phenotype out of 20 to 50 females of the following genotypes: w^m4h/w¹¹¹⁸; Pcif1^M6stop/+ or w^m4h/w¹¹¹⁸; Pcif1^F9ΔNLS/+ compared to control w^m4h/w¹¹¹⁸; +/+ flies (first row) and w^m4h/w¹¹¹⁸; D(3L)/+ female flies carrying a chromosomal deletion encompassing the Pcif1 gene [Df(3L)BSC563, Df(3L)BSC797, or Df(3L)BSC452 as indicated on figure] compared to w^m4h/w¹¹¹⁸; +/+ flies (second row). (C) Quantification of pigment absorbance was performed for five heads per genotype at a wavelength of 480 nm. Ten replicates were performed. One representative experiment out of two is shown. One-way ANOVA test was performed using GraphPad Prism9. P-value: (****) P < 0.0001.