LincRNAs enable germ cells differentiation by promoting PUF proteins condensation

Background:

Proliferative germline stem cells can differentiate into gametes, which are essential for reproduction (Li & Xie, 2005). The overall organization of the C. elegans germline is similar to that in other organisms, containing self-renewing stem cells at the distal end and mature gametes at the proximal end, with germ cells at various stages of differentiation in between. The distal germline can generally be divided into three zones: early progenitor zone (early PZ), late progenitor zone (late PZ) and leptotene/zygotene zone (LZ).  The stem cell niche is formed and kept at the early PZ by the distal tip cell (DTC) through the activation of the conserved GLP-1/Notch signaling pathway. The early PZ consists of stem cells, while the late PZ contains stem cells as well as the meiotic S-phase cells. As the cells enter the LZ, they enter the first stages of meiosis, undergoing a proliferation-to-differentiation transition (Albert & Schedl, 2019) (Figure 1).

Figure 1: C. elegans germline organization. Taken from Falk et al. Figure 1.

Two members of the conserved PUF family of RNA-binding proteins, FBF-1 and 2, are the major regulators of germline stem cell development in C. elegans. The FBFs primarily repress gene expression in the germline by destabilizing target transcripts or blocking translation. Among FBF targets are cell cycle regulators and several long intervening ncRNAs (lincRNAs) (Nishanth & Simon, 2019). Inactivating fbf-1 or fbf-2 alone does not significantly affect fertility; however, the double mutant is sterile because of a disruption in stem cell maintenance, demonstrating the crucial roles of the PUF proteins in regulating stem cell fate (Crittenden et al., 2002).

Although FBFs share many targets and perform overlapping functions, they have opposing effects on germline stem cell dynamics.  FBF-1 facilitates meiotic mRNA degradation and/or transport from the stem cell region, whereas FBF-2 primarily inhibits translation (Voronina et al., 2012). It was previously proposed that the FBFs are expressed in a gradient such that high FBF levels in the early PZ promote a “mitotic” or undifferentiated cell fate and that their expression drop in late PZ and LZ relieves the repression of meiosis-promoting mRNAs (Byrd & Kimble, 2009). Curiously, however, FBF proteins are found in late PZ and LZ (Chen et al., 2012) (see more below), suggesting that their activity, rather than expression, is inhibited at these stages through a yet unknown mechanism that enables germ cell differentiation.

In this preprint, Falk and colleagues could show that three lincRNAs, linc-4, linc-29, and linc-7, which are known to bind to FBF-1 and -2, control meiotic entry and promote germ cell differentiation by sequestering FBFs in germline P granules (perinuclear RNA-protein condensates), thereby blocking their pro-mitotic functions.

Key Findings:

• FBF-1 & FBF-2 are expressed in early meiotic prophase.

In contrast to the “gradient model” which posits that differential expression of FBF proteins controls the mitosis/miosis decision (Byrd & Kimble, 2009), this preprint and a previous study (Chen et al., 2012) found that the FBFs are present throughout the distal germline, with the highest expression in the PZ. While FBF-1 levels drop modestly in the LZ, FBF-2 levels remain high in all zones. Hence, the authors hypothesized that FBFs are suppressed in cells undergoing miosis, perhaps by long non-coding RNAs, which have previously been shown to inhibit the actions of PUF proteins in mammals.

• Three lincRNAs interact with FBFs and inhibit their pro-mitotic functions.

A previous study identified six lincRNAs that are highly expressed in the C. elegans germline. Inactivating individual lincRNAs did not cause any obvious fertility defects (Ishtayeh et al., 2020). Three of the lincRNAs, linc-4, linc-7, and linc-29, have previously been shown to bind the FBFs (Prasad et al., 2016). In this study, the authors found that deleting either linc-4 or linc-7 alone caused a very modest reduction in brood size, while removing both lincRNAs simultaneously led to a significant decrease in fertility. Similarly, a linc-29 single mutant showed only a small drop in brood size whereas the linc-4; linc-29; linc-7 triple deletion mutant (3xlnc) had the smallest brood size compared to WT and the double mutants.

To test whether the FBF-binding ability of lincRNAs is important for fertility, the authors deleted the FBF binding element (FBE) of linc-7. Removing FBE in linc-7 in a linc-4; linc-29 mutant background significantly reduced brood size, phenocopying 3xInc. Together with the findings that linc-4 and -7 are upregulated in meiotic germ cells, the authors put forth a model in which the binding of lincRNAs to FBFs suppresses their activity, promoting meiosis.

• lincRNAs antagonize FBF-2, but not FBF-1, to promote meiosis.

By examining the number of nuclei and mitotic cells in the PZ, the authors found that 3xlnc had a similar effect as fbf-1(-) such that both showed a smaller number of PZ nuclei and a higher mitotic index than WT. Removing fbf-1 in the 3xlnc mutant further exacerbated this phenotype. On the other hand, removing fbf-2 led to an increase in PZ nuclei. Furthermore, inactivating the three lincRNAs in fbf-2(-) did not enhance its phenotype, suggesting that linc-4, linc-29, and linc-7 are epistatic to FBF-2.

Next, the authors performed RNA-seq analysis on WT, the fbf-2(-) and the 3xlnc mutants. Strikingly, they found that 102 of the 221 downregulated genes in 3xlnc physically interact with FBF-2, suggesting that the three lincRNAs normally antagonize the repressive effects of FBF-2 on germline genes.

• FBF-2 condensation paves the way to meiosis.

Next, the authors investigated the cellular localization of the lincRNAs using smFISH, focusing on linc-7. They observed expression of linc-7 in perinuclear P granules and nucleoplasm. They also noticed that the signal increased and became more perinuclear as the germ cells entered the LZ, where meiosis starts. Additionally, the authors found that linc-7 almost exclusively co-localized with an FBF-2 reporter in the perinuclear condensate, especially in the germ cells in the late PZ and LZ.

Finally, the authors tested whether the lincRNAs are important for FBF-2 condensation. Indeed, removing linc-4, -29, and -7, led to a reduction of FBF-2 perinuclear condensates; instead, FBF-2 localized to the cytoplasm. Together, these results suggest that lincRNAs sequester FBF-2 in the P granules, thereby promoting entry to meiosis and germ cell differentiation.

What I liked about this preprint:

This preprint closes the gap on how exactly the entrance to meiosis occurs in the C. elegans germline. I liked this preprint because I think that non-coding RNAs have many undiscovered roles during development as elucidated here. It was clever to think that proteins like FBFs, which can bind RNAs, can also be regulated by (nc)RNAs.

Questions to the authors:

• As shown in supplementary figure 2, the linc-29 expression decreases towards LZ, which is not the case with linc-4 and linc-7, yet its deletion causes less fertile worms. Could linc-29 have another mechanism of action instead of decreasing FBF-2 condensation? Have you also checked the single mutants’ levels of FBF-2 condensation?
• What might regulate lincRNA levels throughout the germline?
• Do FBFs have any roles in downstream gamete differentiation? Why are they inhibited rather than degraded?

References

Albert, J., & Schedl, T. (2019). Biology of the Caenorhabditis elegans Germline Stem Cell System. Genetics, 213(4), 1145–1188. https://doi.org/10.1534/genetics.119.300238

Byrd, D. T., & Kimble, J. (2009). Scratching the niche that controls Caenorhabditis elegans germline stem cells. Seminars in Cell & Developmental Biology, 20(9), 1107–1113. https://doi.org/10.1016/j.semcdb.2009.09.005

Chen, D., Zheng, W., Ai, L., Uyhazi, K. E., Zhao, H., & Lin, H. (2012). Pumilio 1 Suppresses Multiple Activators of p53 to Safeguard Spermatogenesis. Current Biology, 22(5), 420–425. https://doi.org/10.1016/j.cub.2012.01.039

Crittenden, S. L., Bernstein, D., Bachorik, J. L., Thompson, B. E., Gallegos, M., Petcherski, A. G., Moulder, G., Barstead, R., Wickens, M., & Kimble, J. (2002). A conserved RNA-binding protein controls germline stem cells in Caenorhabditis elegans. Nature, 417(6889), 660–663. https://doi.org/10.1038/nature754

Ishtayeh, H., Achache, H., Kroizer, E., Rappaport, Y., Itskovits, E., Gingold, H., Best, C., Rechavi, O., & Tzur, Y. B. (2020). Systematic analysis of long intergenic non-coding RNAs in C. elegans germline uncovers roles in somatic growth. RNA Biology, 18(3), 435–445. https://doi.org/10.1080/15476286.2020.1814549

Li, L., & Xie, T. (2005). STEM CELL NICHE: Structure and Function. Annual Review of Cell and Developmental Biology, 21(1), 605–631. https://doi.org/10.1146/annurev.cellbio.21.012704.131525

Nishanth, M. J., & Simon, B. (2019). Functions, mechanisms and regulation of Pumilio/Puf family RNA binding proteins: a comprehensive review. Molecular Biology Reports, 47(1), 785–807. https://doi.org/10.1007/s11033-019-05142-6

Prasad, A., Porter, D. F., Kroll-Conner, P., Mohanty, I., Ryan, A. R., Crittenden, S. L., Wickens, M., & Kimble, J. (2016). The PUF binding landscape in metazoan germ cells. RNA, 22(7), 1026–1043. https://doi.org/10.1261/rna.055871.116

Voronina, E., Paix, A., & Seydoux, G. (2012). The P granule component PGL-1 promotes the localization and silencing activity of the PUF protein FBF-2 in germline stem cells. Development, 139(20), 3732–3740. https://doi.org/10.1242/dev.083980

Tags: c. elegans, germline, meiosis, mitosis, stem cell, stemness, worm

doi: https://doi.org/10.1242/prelights.35857

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