Germline Stem Cells Differentiation is Attenuated by Long Non-coding Rnas

The transition from proliferative germline stem-cells into differentiating meiocytes has been extensively studied, yet our knowledge of the factors which control this critical process is limited. In the nematode C. elegans the cells are organized in a developmental-spatial manner, from the stem-cells to the mature oocyte, providing the perfect setup to answer this question. Two evolutionary-conserved PUF proteins inhibit the expression of hundreds of genes to maintain stem-cell proliferation, but how differentiation initiates is still unknown since the PUFs are present at high-levels within differentiating meiocytes.

We identified three long intergenic-non-coding RNAs (lincRNAs) which bind the PUF proteins. LincRNAs are transcripts longer than 200 that are not translated into proteins. We engineered CRISPR-Cas-9 lincRNA deletion strains and found that individual deletions lead to small reductions in fertility, whereas double and triple mutant strains lead to dramatic reduction in fertility. Immunofluorescence showed that in gonads of the triple lincRNA mutant there are less mitotic and proliferative cells. Using transcriptomic analyses, we found that among genes which are downregulated in the triple lincRNA deletion worms, there is a significant number of genes which are downregulated by the PUF proteins. This suggests that the lincRNAs inhibit the PUFs. In the triple strain there is a reduction in the association of the PUF proteins to germ granules, an evolutionary-conserved phase separated aggregates, in which the action of many proteins is prohibited. We propose that three lincRNAs promote oogenesis by sequestering the PUF proteins into phase separated granules and increasing the expression of meiosis-promoting genes