RNA in formation and regulation of transcriptional condensates

Macrosopic membraneless organelles containing RNA such as the nucleoli, germ granules and the Cajal body have been known for decades. These biomolecular condensates are liquid-like bodies that can form by a phase transition. Recent evidence has revealed the presence of similar microscopic condensates associated with transcription of genes. This brief article summarizes thoughts about the importance of condensates in regulation of transcription and how RNA molecules, as components of such condensates, control the synthesis of RNA. Models and experimental data suggest that RNAs from enhancers facilitate the formation of a condensate that stabilizes the binding of transcription factors and accounts for a burst of transcription at the promoter. Termination of this burst is pictured as a non-equilibrium feedback loop where additional RNA destabilizes the condensate.

Proximity labeling identifies LOTUS domain proteins that promote the formation of perinuclear germ granules in C. elegans

The germ line produces gametes that transmit genetic and epigenetic information to the next generation. Maintenance of germ cells and development of gametes require germ granules-well-conserved membraneless and RNA-rich organelles. The composition of germ granules is elusive owing to their dynamic nature and their exclusive expression in the germ line. Using C. elegans germ granule, called P granule, as a model system, we employed a proximity-based labeling method in combination with mass spectrometry to comprehensively define its protein components. This set of experiments identified over 200 proteins, many of which contain intrinsically disordered regions. An RNAi-based screen identified factors that are essential for P granule assembly, notably EGGD-1 and EGGD-2, two putative LOTUS-domain proteins. Loss of eggd-1 and eggd-2 results in separation of P granules from the nuclear envelope, germline atrophy and reduced fertility. We show that intrinsically disordered regions of EGGD-1 are required to anchor EGGD-1 to the nuclear periphery while its LOTUS domains are required to promote perinuclear localization of P granules. Together, our work expands the repertoire of P granule constituents and provides new insights into the role of LOTUS-domain proteins in germ granule organization.

Distinct phases within large RNP granules of C. elegans oocytes are associated with differential stress responses of RNA binding proteins

One emerging paradigm of cellular organization of RNA and RNA binding proteins is the formation of membraneless organelles (MLOs). Examples of MLOs include several types of RNP (ribonucleoprotein) granules that form via phase separation. Proper regulation of the phase transitions of RNA binding proteins is critical as dysregulation can lead to disease states. Germ granules are small RNP granules conserved across metazoa. In C. elegans, when oogenesis undergoes an extended meiotic arrest, germ granules assemble into much larger, more complex RNP granules whose hypothesized function is to regulate RNA metabolism and maintain oocyte quality. As a step towards gaining insight into the function of RNP granules, in this report we characterize distinct phases for four RNA binding proteins in arrested oocytes. We find that PGL-1 is dynamic and has liquid-like properties, while MEG-3 has gel-like properties, both similar to their properties in small germ granules of embryos. We also show that MEX-3 exhibits gel-like properties in many regards but is more dynamic than MEG-3. We find CGH-1 is dynamic but does not consistently behave liquid-like, and may be an intermediate phase within RNP granules. We also show that the distinct phases of the RNA binding proteins are associated with differential responses to stress.

Tools to Image Germplasm Dynamics During Early Zebrafish Development

During the first day of zebrafish development, ribonucleoprotein (RNP) complexes called germplasm form large aggregates that initially segregate asymmetrically during cleavage stages. After zygotic genome activation, the granules break into smaller fragments that associate with the nuclear membrane as perinuclear (germ) granules toward the end of gastrulation. The mechanisms underlying the highly dynamic behavior of germ granules are not well studied but thought to be facilitated by the cytoskeleton. Here, we present efficient mounting strategies using 3d-printed tools that generate wells on agarose-coated sample holders to allow high-resolution imaging of multiplexed embryos that are less than one day post-fertilization (dpf) on inverted (spinning disk confocal) as well as upright (lattice light-sheet and diSPIM) microscopes. In particular, our tools and methodology allow water dipping lenses to have direct access to mounted embryos, with no obstructions to the light path (e.g., through low melting agarose or methyl cellulose). Moreover, the multiplexed tight arrays of wells generated by our tools facilitate efficient mounting of early embryos (including cleavage stages) for live imaging. These methods and tools, together with new transgenic reporter lines, can facilitate the study of germ granule dynamics throughout their lifetime in detail, at high resolution and throughput, using live imaging technologies.

Proximity labeling identifies LOTUS domain proteins that promote the formation of perinuclear germ granules in C. elegans

The germline produces gametes that transmit genetic and epigenetic information to the next generation. Maintenance of germ cells and development of gametes require germ granules—well-conserved membraneless and RNA-rich organelles. The composition of germ granules is elusive owing to their dynamic nature and their exclusive expression in the germline. Using C. elegans germ granule, called P granule, as a model system, we employed a proximity-based labeling method in combination with mass spectrometry to comprehensively define its protein components. This set of experiments identified over 200 proteins, many of which contain intrinsically disordered regions. An RNAi-based screen identified factors that are essential for P granule assembly, notably EGGD-1 and EGGD-2, two previously uncharacterized LOTUS-domain proteins. Loss of eggd-1 and eggd-2 results in separation of P granules from nuclear envelope, germline atrophy and reduced fertility. We show that intrinsically disordered regions of EGGD-1 are required to anchor EGGD-1 to the nuclear periphery while its LOTUS domains are required to promote perinuclear localization of P granules. Together, our work expands the repertoire of P granule constituents and provides new insights into the role of LOTUS-domain proteins in germ granule organization.

The Caenorhabditis elegans TDRD5/7-like protein, LOTR-1, interacts with the helicase ZNFX-1 to balance epigenetic signals in the germline

LOTUS and Tudor domain containing proteins have critical roles in the germline. Proteins that contain these domains, such as Tejas/Tapas in Drosophila, help localize Vasa to the germ granules and facilitate piRNA-mediated transposon silencing. The homologous proteins in mammals, TDRD5 and TDRD7, are required during spermiogenesis. Until now, proteins containing both LOTUS and Tudor domains in Caenorhabditis elegans have remained elusive. Here we describe LOTR-1 (D1081.7), which derives its name from its LOTUS and Tudor domains. Interestingly, LOTR-1 docks next to P granules to colocalize with the broadly conserved Z-granule helicase, ZNFX-1. LOTR-1's Z-granule association requires its Tudor domain, but both LOTUS and Tudor deletions affect brood size when coupled with a knockdown of the Vasa homolog glh-1. In addition to interacting with the germ-granule components WAGO-1, PRG-1 and DEPS-1, we identified a Tudor-dependent association with ZNFX-1. Like znfx-1 mutants, lotr-1 mutants lose small RNAs from the 3' ends of WAGO and Mutator targets, reminiscent of the loss of piRNAs from the 3' ends of piRNA precursor transcripts in mouse Tdrd5 mutants. Our work suggests that LOTR-1 acts in a conserved mechanism that brings small RNA generating mechanisms towards the 3' ends of small RNA templates or precursors.

Protein-based condensation mechanisms drive the assembly of RNA-rich P granules

Germ granules are protein-RNA condensates that segregate with the embryonic germline. In C. elegans embryos, germ (P) granule assembly requires MEG-3, an intrinsically-disordered protein that forms RNA-rich condensates on the surface of PGL condensates at the core of P granules. MEG-3 is related to the GCNA family and contains an N-terminal disordered region (IDR) and a predicted ordered C-terminus featuring an HMG-like motif (HMGL). We find that MEG-3 is modular protein that uses its IDR to bind RNA and its C-terminus to drive condensation. The HMGL motif mediates binding to PGL-3 and is required for co-assembly of MEG-3 and PGL-3 condensates in vivo. Mutations in HMGL cause MEG-3 and PGL-3 to form separate condensates that no longer co-segregate to the germline or recruit RNA. Our findings highlight the importance of protein-based condensation mechanisms and condensate-condensate interactions in the assembly of RNA-rich germ granules.

Translation and codon usage regulate Argonaute slicer activity to trigger small RNA biogenesis

In the Caenorhabditis elegans germline, thousands of mRNAs are concomitantly expressed with antisense 22G-RNAs, which are loaded into the Argonaute CSR-1. Despite their essential functions for animal fertility and embryonic development, how CSR-1 22G-RNAs are produced remains unknown. Here, we show that CSR-1 slicer activity is primarily involved in triggering the synthesis of small RNAs on the coding sequences of germline mRNAs and post-transcriptionally regulates a fraction of targets. CSR-1-cleaved mRNAs prime the RNA-dependent RNA polymerase, EGO-1, to synthesize 22G-RNAs in phase with translating ribosomes, in contrast to other 22G-RNAs mostly synthesized in germ granules. Moreover, codon optimality and efficient translation antagonize CSR-1 slicing and 22G-RNAs biogenesis. We propose that codon usage differences encoded into mRNA sequences might be a conserved strategy in eukaryotes to regulate small RNA biogenesis and Argonaute targeting.