Fig. 2
From: Activity dependent LoNA regulates translation by coordinating rRNA transcription and methylation
LoNA coordinates rRNA transcription and protein translation. a Nucleolar localization of LoNA. Representative immunofluorescence images of polR1E (red), LoNA RNA FISH (green), and DAPI (blue). Scale bar: 10 µm. b LoNA lacks m2,2,7G and m7G cap. IP was performed with total RNAs isolated from N2a cells using anti-m2,2,7G/m7G antibody or control IgG, followed by qPCR detection. Bar plots represent fold enrichments of immunoprecipitated RNAs by antibody over IgG control. U1 snRNA, U3 snoRNA, and FBL were included as positive controls. c LoNA is polyadenylated. poly (A)+ and poly (A)− RNAs were purified from N2a total RNAs by oligo(dT) beads pulldown, followed by qPCR. Data were presented as relative abundance of a particular RNA in each pool. GAPDH was included as poly (A)+ control, U1 snRNA as poly (A)− control. d–g Levels of mature rRNAs (28S, 18S, 5.8S, and 5S) were determined by Northern blot (U1 was used as control), densitometric analysis, and qPCR (data were normalized to U1 snRNA) in LoNA-overexpressed N2a cells (LoNA OV) (d–e), or LoNA knockdown N2a cells (LoNA KD) (f, g). h Nascent pre-rRNA (45S) levels were determined by nuclear run-on assay. i–l Levels of pre-rRNA (45S) and intermediate rRNAs (41S, 36S, and 34S) were determined in LoNA transfected N2a cells (LoNA OV) by Northern blot (U1 was used as control), densitometric analysis (i) and qPCR (45S only) (e), as well as in LoNA-deficient N2a cells (LoNA KD) by Northern blot (U1 was used as control), densitometric analysis (k) and qPCR (45S only) (g). j, l Ratio of total intermediate rRNA to the precursor 45S, as determined by densitometric analysis. m, n LoNA administered or deficient N2a cells were pulsed with 35S-Met/Cys, de novo protein synthesis was quantified as the mean ratio of 35S incorporation relative to total protein (Coomassie). For this and subsequent figures, OV represents overexpression, KD represents knockdown. Sequences of Northern blot probes and qPCR primers are listed in supplementary materials. *P < 0.05; **P < 0.01; ***P < 0.001 by ANOVA or two-tailed Student’s t test, error bars, s.e.m.
