PRMT5 and the role of symmetrical dimethylarginine in chromatoid bodies of planarian stem cells

Abstract

Planarian flatworms contain a population of adult stem cells (neoblasts) that proliferate and generate cells of all tissues during growth, regeneration and tissue homeostasis. A characteristic feature of neoblasts is the presence of chromatoid bodies, large cytoplasmic ribonucleoprotein (RNP) granules morphologically similar to structures present in the germline of many organisms. This study aims to reveal the function, and identify additional components, of planarian chromatoid bodies. We uncover the presence of symmetrical dimethylarginine (sDMA) on chromatoid body components and identify the ortholog of protein arginine methyltransferase PRMT5 as the enzyme responsible for sDMA modification in these proteins. RNA interference-mediated depletion of planarian PRMT5 results in defects in homeostasis and regeneration, reduced animal size, reduced number of neoblasts, fewer chromatoid bodies and increased levels of transposon and repetitive-element transcripts. Our results suggest that PIWI family member SMEDWI-3 is one sDMA-containing chromatoid body protein for which methylation depends on PRMT5. Additionally, we discover an RNA localized to chromatoid bodies, germinal histone H4. Our results reveal new components of chromatoid bodies and their function in planarian stem cells, and also support emerging studies indicative of sDMA function in stabilization of RNP granules and the Piwi-interacting RNA pathway.

PIWI homologs mediate Histone H4 mRNA localization to planarian chromatoid bodies

“…To examine whether these histone mRNAs also localize to chromatoid bodies, we performed FISH combined with immunolabeling of chromatoid bodies using the Y12 antibody as described previously (Fig. 1) (Rouhana et al, 2012). Indeed, we observed enrichment in chromatoid bodies for transcripts corresponding to core Histone H2A and H3 (∼74% and 56% of chromatoid bodies, respectively), and to a lesser extent for transcripts corresponding to Histone H1 (∼40%) and H2B (∼34%) mRNAs ( Fig.…”

“…This sequence was named germinal histone H4 (gH4) and has been used as a marker for planarian neoblasts and germ cells (Collins et al, 2010;Wang et al, 2010Wang et al, , 2007. Unlike many other neoblast-enriched transcripts, gH4 transcripts localize to chromatoid bodies (Rouhana et al, 2012), as judged by colocalization of fluorescent in situ hybridization (FISH) signals with Y12 antibody (Lerner et al, 1981) labeling of chromatoid bodies ( Fig. 1A,B; supplementary material Fig.…”

“…S1). Y12 binds specifically to symmetrical dimethylarginine (sDMA), a posttranslational modification that has been detected in RNAprocessing factors, such as Sm proteins (Brahms et al, 2000) and PIWI homologs (for full characterization, see Rouhana et al, 2012). It is important to note that gH4 mRNA is also observed away from chromatoid bodies in the cytoplasm of neoblasts (Fig.…”

“…Of these, sequence with the greatest identity to gH4 corresponds to a locus that contains a gH4 sequence duplication, flanking transposon remnants and piRNA islands (Rouhana et al, 2012). The transcript that resulted in the gH4 cDNA clone used in our studies is predicted to originate from a spliced and polyadenylated product from this locus, which contains a long 3′UTR (supplementary material Fig.…”

“…In planarians, these membrane-less organelles are known as 'chromatoid bodies', and their function has remained elusive for decades (Auladell et al, 1993;Coward, 1974;Hori, 1982;Morita et al, 1969). Planarian chromatoid bodies contain homologs of the RNA helicase DHH1/RCK (Yoshida-Kashikawa et al, 2007), Tudor (Solana et al, 2009), SmB (Fernandez-Taboada et al, 2010) and methylated substrates of the protein arginine methyltransferase 5 (PRMT5) (Rouhana et al, 2012). PRMT5 catalyzes the synthesis of symmetrical dimethylated arginines (sDMA) on SmB, Vasa and PIWI homologs (Blackwell and Ceman, 2012).…”