GARBARINO PICO EDUARDO
Congresos y reuniones científicas
Título:
Subcellular localization of the circadian deadenylase nocturnin
Autor/es:
GARBARINO PICO E; GREEN CB
Lugar:
Destin, Florida
Reunión:
Congreso; XI Meeting Society for Research on Biological Rhythms; 2008
Institución organizadora:
Society for Research on Biological Rhythms
Resumen:
Nocturnin (Noc) is a rhythmic deadenylase (polyA-specific ribonuclease) that in mouse is expressed in multiple tissues at night. Deadenylation induces silencing or degradation in most transcripts. Our hypothesis is that NOC is a posttranscriptional regulator of the rhythmic expression of some circadian-related mRNAs. Here we asked whether mNOC localizes in cell subdomains where ribonucleoprotein complexes (RNPs) are processed.
We expressed mNOC with two different tags: a C-term FLAG and an N-term GFP. Both constructions localized in the cytoplasm of NIH3T3 and HEK293 cells showing a diffuse signal, and in some cases, a few perinuclear dots. We then assessed by ICC whether mNOC co-localizes with markers of stress granules (SGs), sites where non-translating mRNAs are stored together with 40S ribosomal subunits, RNA-binding proteins (RBPs), some translational factors, and other proteins. mNOC did not colocalize with either of two SG markers. Next, we tested whether mNOC is in processing bodies (PBs), places where factors involved in 5? to 3? mRNA decay, RBPs, and other mRNA processing proteins are concentrated. mNOC-FLAG was not detectable in these structures. Finally, polysome analyses indicated that endogenous mNOC is not associated with ribosomes or polyribosomes.
Our results show that mNOC localizes in the cytoplasm, outside of SGs, PBs, and polyribosomes. However we cannot rule out the possibility that mNOC translocates to some of those structures in other conditions or cell types. In addition, novel cytoplasmic subdomains where RNPs are remodeled have been described, and could represent the dots seen in the mNOC staining.
We expressed mNOC with two different tags: a C-term FLAG and an N-term GFP. Both constructions localized in the cytoplasm of NIH3T3 and HEK293 cells showing a diffuse signal, and in some cases, a few perinuclear dots. We then assessed by ICC whether mNOC co-localizes with markers of stress granules (SGs), sites where non-translating mRNAs are stored together with 40S ribosomal subunits, RNA-binding proteins (RBPs), some translational factors, and other proteins. mNOC did not colocalize with either of two SG markers. Next, we tested whether mNOC is in processing bodies (PBs), places where factors involved in 5? to 3? mRNA decay, RBPs, and other mRNA processing proteins are concentrated. mNOC-FLAG was not detectable in these structures. Finally, polysome analyses indicated that endogenous mNOC is not associated with ribosomes or polyribosomes.
Our results show that mNOC localizes in the cytoplasm, outside of SGs, PBs, and polyribosomes. However we cannot rule out the possibility that mNOC translocates to some of those structures in other conditions or cell types. In addition, novel cytoplasmic subdomains where RNPs are remodeled have been described, and could represent the dots seen in the mNOC staining.
