mRNA localization is linked to translation regulation in the Caenorhabditis elegans germ lineage

ABSTRACT Caenorhabditis elegans early embryos generate cell-specific transcriptomes despite lacking active transcription, thereby presenting an opportunity to study mechanisms of post-transcriptional regulatory control. We observed that some cell-specific mRNAs accumulate non-homogenously within cells, localizing to membranes, P granules (associated with progenitor germ cells in the P lineage) and P-bodies (associated with RNA processing). The subcellular distribution of transcripts differed in their dependence on 3′UTRs and RNA binding proteins, suggesting diverse regulatory mechanisms. Notably, we found strong but imperfect correlations between low translational status and P granule localization within the progenitor germ lineage. By uncoupling translation from mRNA localization, we untangled a long-standing question: Are mRNAs directed to P granules to be translationally repressed, or do they accumulate there as a consequence of this repression? We found that translational repression preceded P granule localization and could occur independently of it. Further, disruption of translation was sufficient to send homogenously distributed mRNAs to P granules. These results implicate transcriptional repression as a means to deliver essential maternal transcripts to the progenitor germ lineage for later translation.

. P 1 -enriched, clustered transcripts co-localize with the P granule marker PGL-1::GFP. In addition to co-localizing with GFP signal in the P granule marker strain containing GLH-1::GFP (Fig. 3), chs-1, clu-1, cpg-2, and nos-2 mRNAs (all in magenta) also co-localize with a second P granule marker protein, PGL-1::GFP (green). DAPI is illustrated in blue to visualize nuclei and illustrate the 4-cell stage of development.  S4. Quantification of mRNA cluster overlap with the P granules. mRNA cluster overlap with GLH-1::GFP labeled P granules is calculated using micrographs of GLH-1::GFP and clustered RNAs (clu-1 shown) (A, left), computationally identifying P granules and RNA clusters (A, middle), and comparing the 3D masks for overlap to identify independent P granules and RNA clusters (magenta) or colocalized clusters (green) (A, right). (B) A Venn-Euler diagram illustrating the number of independent clu-1 mRNA clusters (magenta), independent P granules (light green), and overlapping P granules and mRNA clusters (dark green) in a single embryo (from A). (C) Box plots comparing the size of non-overlapping mRNA clusters and P granules to those overlapping shows larger mRNA clusters more commonly overlap with P granules (left) and brighter P granules more commonly overlap with mRNA clusters (right).

Fig. S5
. Immunofluorescence control images for anti-GFP antibody staining. The anti-GFP antibody reports PATR-1::GFP localization (green, right) in PATR-1::GFP containing strains as compared to N2 wild type un-transformed control strains (green, left). DAPI staining is shown in blue to visualize nuclei and illustrate the 4-cell stage of development.

Fig. S6
. mRNA clusters display homotypic clustering within P granules. chs-1 mRNA (magenta) tend to homotypically cluster in the core of P granules while clu-1 mRNA (green) also cluster homotypically, but near the peripheries of P granules.

Fig. S13. Upon heat shock, a greater number of mRNAs clusters co-localize with P granules.
Quantification of smFISH assays of B0495.7, gpd-2, and set-3, three transcripts that are typically not P granule localized. (A) Upon heat shock (30º C), larger and more numerous clusters formed. (B) Upon heat shock, more numerous RNA clusters co-localized with the P granule marker protein (GLH-1::GFP). The percentages of clusters overlapping P granule markers are shown in Figure 7B. Here, the raw numbers of co-localized clusters are tabulated. Statistics were performed using Welch's Two Sample t-test p-values: 0.05 > * ≥ 0.005; 0.005 > ** ≥ 0.0005; *** < 0.0005. Table S1. Worm strains used in this study. Table S2. E.coli strains and plasmids used in this study. Table S3. Oligonucleotide primers used in this study. Table S4. smFISH and smiFISH probesets used in this study.
Click here to Download Table S1 Click here to Download Table S2 Click here to Download Table S3 Click here to Download Table S4