Comprehensive analysis of nucleocytoplasmic dynamics of mRNA in Drosophila cells

Abstract

Eukaryotic mRNAs undergo a cycle of transcription, nuclear export, and degradation. A major challenge is to obtain a global, quantitative view of these processes. Here we measured the genome-wide nucleocytoplasmic dynamics of mRNA in Drosophila cells by metabolic labeling in combination with cellular fractionation. By mathematical modeling of these data we determined rates of transcription, export and cytoplasmic decay for 5420 genes. We characterized these kinetic rates and investigated links with mRNA features, RNA-binding proteins (RBPs) and chromatin states. We found prominent correlations between mRNA decay rate and transcript size, while nuclear export rates are linked to the size of the 3'UTR. Transcription, export and decay rates are each associated with distinct spectra of RBPs. Specific classes of genes, such as those encoding cytoplasmic ribosomal proteins, exhibit characteristic combinations of rate constants, suggesting modular control. Binding of splicing factors is associated with faster rates of export, and our data suggest coordinated regulation of nuclear export of specific functional classes of genes. Finally, correlations between rate constants suggest global coordination between the three processes. Our approach provides insights into the genome-wide nucleocytoplasmic kinetics of mRNA and should be generally applicable to other cell systems.

Fig 1. Mathematical modeling of the nucleocytoplasmic dynamics of mRNA.

(A) Schematic illustration of the kinetic steps in the model. (B, C) Fitting of the model to experimental data for two example genes (Arc1 and Bacc). Green dots and blue dots represent nuclear and cytoplasmic transcript abundance, respectively, normalized to yeast spike-in. Red curves depict the fitted kinetic model. (D, E) Global distribution of the goodness of fit scores for nuclear and cytoplasmic fractions, respectively, assessed by coefficient of determination (r²). Only genes with r² > 0.8 are used for downstream analyses. (F, G, H) Scatter plots showing the reproducibility of modeled rates of transcription, export and cytoplasmic decay of two biological replicates. Each dot represents one gene. Red lines indicate the perfect diagonals. (I) Contributions of the variance of the rates of transcription, export and cytoplasmic decay to the variance of steady state transcript abundance.

Fig 2. The relationships between transcript length and kinetic rates of mRNA.

(A, B, C) Scatterplots showing the relationships between transcript length, intron length, number of exons and transcription rates on logarithmic scales. Red lines indicate fitting by linear regression. Spearman (ρ) correlations are indicated. (D) Relationship of 3’UTR length and export rates. (E, F) Correlations between transcript and 3’UTR length, respectively, and decay rates.

Fig 3. Links between binding of RBPs and kinetic rates of mRNA.

(A) Log₂-transformed ratios of median transcription rates of genes bound ($\widetilde{bd}$) and unbound ($\widetilde{ubd}$) by RBPs as indicated. (B) Log₂-transformed mean Pol II pausing indices of transcripts bound by each RBP ($\overline{bd}$). (C) Same as (A), but for export rates. RBPs with roles in splicing are marked by "x". (D) Same as (A), but for decay rates. RBPs in all the panels are ranked according to their associated values, but colors are same as in (A). RBP binding data was taken from [31]. RBPs of which binding is significantly associated with rates of transcription (A), pausing index (B), export (C) and cytoplasmic decay (D) are marked by (*) (P < 0.01, two-sided Wilcoxon test, adjusted by the Holm—Bonferroni method).

Fig 4. Associations of chromatin states and kinetic rates of mRNA.

Links between chromatin states and transcription rates: (A) transcription rates of genes divided by their chromatin states at their transcription start sites (TSS). Whiskers represent the 5th percentile and the 95th percentile; (B) Between each pair of chromatin states at TSS, a line is drawn if there is statistical difference (p <0.01, Tukey’s range test, ANOVA); (C, D) same as (A, B), but for chromatin states at transcription termination sites (TTS). Similar analyses are displayed for nuclear export (E-H) and cytoplasmic decay (I-L).

Fig 5. Links between kinetic rates; and GO analysis.

(A, B, C) Scatterplots showing pairwise relationships between the rates of transcription, export and cytoplasmic decay on logarithmic scales. Spearman (ρ) coefficients are indicated. Red lines indicate fitting by linear regression. Genes encoding cytoplasmic ribosomal proteins (cRP) are marked in blue and genes encoding mitochondrial ribosomal proteins (mRP) are marked in green. (D) Gene ontology analysis. Heatmap showing associations between kinetic rates and "biological process" GO categories. Processes are shown if they are enriched for at least one kinetic rate with p < 10⁻¹¹. The enrichment scores of genes with the respective GO terms are indicated, with log₁₀-transformed p-values in parentheses, according to GOrilla [82]. Red color marks enrichment for high kinetic rates while blue color marks enrichment for low kinetic rates. See also S2 Table.