PGL germ granule assembly protein is a base-specific, single-stranded RNase

Abstract

Cellular RNA-protein (RNP) granules are ubiquitous and have fundamental roles in biology and RNA metabolism, but the molecular basis of their structure, assembly, and function is poorly understood. Using nematode "P-granules" as a paradigm, we focus on the PGL granule scaffold protein to gain molecular insights into RNP granule structure and assembly. We first identify a PGL dimerization domain (DD) and determine its crystal structure. PGL-1 DD has a novel 13 α-helix fold that creates a positively charged channel as a homodimer. We investigate its capacity to bind RNA and discover unexpectedly that PGL-1 DD is a guanosine-specific, singlestranded endonuclease. Discovery of the PGL homodimer, together with previous results, suggests a model in which the PGL DD dimer forms a fundamental building block for P-granule assembly. Discovery of the PGL RNase activity expands the role of RNP granule assembly proteins to include enzymatic activity in addition to their job as structural scaffolds.C ytoplasmic RNA-protein (RNP) granules are found in virtually all cells and are thought to be central to RNA metabolism (1, 2). These diverse organelles include P-bodies, stress granules, neuronal granules, and germ granules (2). RNP granules are not membrane-bound and display liquid-liquid phase-separation properties (3, 4). Many of their molecular components have been identified, including scaffold proteins: proteins that recruit other key granule components and are sufficient to induce RNP granule assembly. Major challenges now are to understand how RNP granules are assembled and how they control RNAs.Germ granules are exemplary RNP granules with a profound yet largely mysterious role in metazoan germ-line development. These granules possess common components across phyla (5) but use unique scaffold proteins, such as Drosophila Oskar (6), zebrafish Bucky Ball (7), and Caenorhabditis elegans paralogs PGL-1 and PGL-3 (8, 9), called PGL collectively. Germ granule scaffold proteins from different phyla have distinct amino acid sequences with no conserved domains. The importance of these scaffolds has been attributed to their function in germ granule assembly (for examples, see refs. 10-12). However, the molecular basis of that assembly and how it impacts RNA regulation remain unknown.Here we focus on the Caenorhabditid PGL scaffold proteins and their role in assembly of nematode germ granules, called P-granules (13). P-granules are required for germ-line survival (8, 9) and germline totipotency (14). A recent model proposes that P-granules capture selected mRNAs exiting the nucleus (15), an idea based on the finding that untranslated mRNAs are enriched in P-granules, but translated mRNAs are absent (15, 16). The consequences of that capture are unclear but may include mRNA repression.The PGL family comprises the closely related PGL-1 and PGL-3 proteins plus divergent PGL-2. PGL-1 and PGL-3 are required for adult germ cell development but the function of PGL-2 is unknown (8). All three PGL proteins interact with each other in vitro ...

Germ Granules Prevent Accumulation of Somatic Transcripts in the AdultCaenorhabditis elegansGermline

“…Indeed, some germline transcripts have been shown to at least transiently reside in P granules (Schisa et al 2001;Sheth et al 2010). Recently, PGL-1 was shown to dimerize and to have guanosinespecific RNase activity (Aoki et al 2016). This raises the possibility that PGL-1 directly influences the transcriptome by cleaving its associated mRNAs, although PGL-1's catalytic activity is not essential for its role in fertility (Aoki et al 2016).…”

“…Recently, PGL-1 was shown to dimerize and to have guanosinespecific RNase activity (Aoki et al 2016). This raises the possibility that PGL-1 directly influences the transcriptome by cleaving its associated mRNAs, although PGL-1's catalytic activity is not essential for its role in fertility (Aoki et al 2016). If PGL-1 does indeed have RNase activity in vivo, then an attractive scenario is that it recognizes mRNAs inappropriate for germline development and cleaves them, while allowing germline-appropriate mRNAs to pass through P granules and be translated in the cytoplasm (Figure 8).…”

Liquid droplet germ granules require assembly and localized regulators for mRNA repression

“…Because PGLs are key assembly proteins for P-granules and can form granules on their own 6, 11, 24, 25 , we reasoned that PGLs use multiple self-interactions to drive granule assembly. We previously identified one dimerization domain (DD) centrally in PGL 27 , but DD missense mutations grossly affected protein stability. We postulated the existence of another PGL multimerization domain that might be more amenable to manipulation and again turned to structural analyses.…”

“…Assays for granule assembly in mammalian cells implicated the conserved N-terminal region of PGL as critical 25 , and structural studies identified a central dimerization domain (DD) within that conserved region ( Figure 1B ) 27 . Yet higher-ordered self-assembly demands additional PGL-PGL contacts.…”

Germ granules prevent accumulation of somatic transcripts in the adultC. elegansgermline

“…Taken together, these findings reveal that C. elegans germ 10 granules are major regulators of the germline transcriptome and prevent germ cells from 11 undergoing somatic development, possibly by preventing the expression of somatic factors in the 12 germline ( Figure 8). The mechanism by which P granules perform these functions remains to be 13 for its role in fertility (Aoki et al 2016). If PGL-1 indeed has RNase activity in vivo, then an 23 attractive scenario is that it recognizes mRNAs inappropriate for germline development and cleaves 24 them, while allowing germline-appropriate mRNAs to pass through P granules and be translated in 25 the cytoplasm (Figure 8).…”