Poly (A)-specific ribonuclease deficiency impacts oogenesis in zebrafish

Abstract

Poly (A)-specific ribonuclease (PARN) is the most important 3'-5'exonuclease involved in the process of deadenylation, the removal of poly (A) tails of mRNAs. Although PARN is primarily known for its role in mRNA stability, recent studies suggest several other functions of PARN including a role in telomere biology, non-coding RNA maturation, trimming of miRNAs, ribosome biogenesis and TP53 function. Moreover, PARN expression is de-regulated in many cancers, including solid tumours and hematopoietic malignancies. To better understand the in vivo role of PARN, we used a zebrafish model to study the physiological consequences of Parn loss-of-function. Exon 19 of the gene, which partially codes for the RNA binding domain of the protein, was targeted for CRISPR-Cas9-directed genome editing. Contrary to the expectations, no developmental defects were observed in the zebrafish with a parn nonsense mutation. Intriguingly, the parn null mutants were viable and fertile, but turned out to only develop into males. Histological analysis of the gonads in the mutants and their wild type siblings revealed a defective maturation of gonadal cells in the parn null mutants. The results of this study highlight yet another emerging function of Parn, i.e., its role in oogenesis.

Figure 1

Generation of parn-KO zebrafish. (a) Pictorial representation of all the exons of parn and highlight of the target region. (b) shows the different domains (CAF1-Chromatin Assembly Factor 1 complex; R3H- Arginine and Histidine; RBD-RNA Binding Domain) of Parn wildtype and Parn -/-. (c) The graph shows the mRNA level of parn homozygous mutant ((parn ^{5∆/ 5∆}) and wildtype (age-1.5 years), p value < 0.0001 was considered statistically significant. The error bar represents the standard deviation (SD), the results were analysed using GraphPad prism 8.4.3.

Figure 2

Representative images of embryos obtained from a parn heterozygous (parn ^5∆/+) cross at 0hpf (a), 6hpf (b), 10hpf (c), 18hpf (d), 24hpf (e), 48hpf (f), 72hpf (g), 96hpf (h) and 120hpf (i) to show that all were phenotypical identical. All images were captured using Leica S9D, Camera MC190.

Figure 3

(a) Representative images of embryos obtained from a parn heterozygous cross at day 3. (b–d) shows the electropherogram of wildtype, heterozygous (parn ^5∆/+) and homozygous (parn ^5∆/5∆) mutant embryos respectively. The region highlighted in ‘b’ shows 5 bases ‘CCTGA’ intact in wildtype. In ‘c’ the arrows represent the rearrangement of bases due to loss of 5 bases ‘CCTGA’ in one allele. The highlighted region in ‘d’ shows the stop codon (TAA) that arises due to loss of 5 bases ‘CCTGA’ in both the allele. (e), (f), (g) corresponds to embryos at day 3 for wildtype, heterozygous and homozygous mutant respectively with no morphological difference. All images were captured using Leica S9D, Camera MC190. Polypeak parser (yosttools.genetics.utah.edu/PolyPeakParser/) software was used to analyse the sequencing data.

Figure 4

(a) Number of mutants obtained across 4 generation. (b) Shows the sex and the zygosity percentage obtained across 4 generation. (c, d) Representative images of adult parn ^5∆/+ ♂, parn ^5∆/+ ♀ and parn ^{5∆/ 5∆} ♂ showing no morphological difference (age-1.5 years). The images (c) and (d) were captured using a DSLR camera (Canon EOS1300D).

Figure 5

Representative sections of ovary tissue stained with hematoxylin and eosin, from wildtype ♀, parn + / + ♀ obtained from parn ^5∆/+ cross and parn ^5∆/+ ♀ obtained from parn ^5∆/+ cross. The oocytes are shown with arrows and the numbers represent oocytes at corresponding stages of development, Pre-vitellogenic stages indicated as I and II; vitellogenic stage indicated as III and post vitellogenic stage indicated as IV (age-1.5 years). The images were captured using Olympus BX53, Camera DP74, Bright field. ht corresponds to the parn heterozygous mutants (parn ^5∆/+).

Figure 6

Representative sections of testis tissue stained with haematoxylin and eosin, from wildtype ♂, PARN + / + ♂ obtained from parn ^5∆/+ , parn ^5∆/+ ♂ obtained from parn ^5∆/+ cross and parn ^{5∆/ 5∆} obtained from parn ^5∆/+ cross. SC: Spermatocytes, SG: Spermatogonia, SZ: Spermatozoa (age-1.5 years). The images were captured using Olympus BX53, Camera DP74, Bright field. ht corresponds to the parn heterozygous mutants and hm corresponds to the parn homozygous mutants.

Figure 7

The graph shows the basal mRNA level of parn in different organs of adult zebrafish. Of all the organs, ovary show higher mRNA transcript when compared to heart, testis, liver, intestine, kidney and brain. (Age-1.5 years), p value < 0.0001 was considered statistically significant. The error bar represents the standard error mean (SEM), the results were analysed using GraphPad prism 8.4.3.