Rapid nuclear deadenylation of mammalian messenger RNA

Abstract

Poly(A) tails protect RNAs from degradation and their deadenylation rates determine RNA stability. Although poly(A) tails are generated in the nucleus, deadenylation of tails has mostly been investigated within the cytoplasm. Here, we combined long-read sequencing with metabolic labeling, splicing inhibition and cell fractionation experiments to quantify, separately, the genesis and trimming of nuclear and cytoplasmic tails in vitro and in vivo. We present evidence for genome-wide, nuclear synthesis of tails longer than 200 nt, which are rapidly shortened after transcription. Our data suggests that rapid deadenylation is a nuclear process, and that different classes of transcripts and even transcript isoforms have distinct nuclear tail lengths. For example, many long-noncoding RNAs retain long poly(A) tails. Modeling deadenylation dynamics predicts nuclear deadenylation about 10 times faster than cytoplasmic deadenylation. In summary, our data suggests that nuclear deadenylation might be a key mechanism for regulating mRNA stability, abundance, and subcellular localization.

Keywords: Cell biology; Molecular mechanism of gene regulation; Molecular physiology.

Graphical abstract

Figure 1

Unspliced RNAs have long poly(A) tails (A) Browser shot example of detected unspliced read in for gene AHSG in Organoid replicate 1 FLAM-seq dataset (number spliced reads = 317; num unspliced = 3). (B) (top) Bulk polyA tail length density distributions for replicates HeLa S3, iPSC, and Organoid datasets in replicates (bottom) Histogram of intronic poly(A) tail length normalized to total reads per dataset for corresponding datasets and replicates. (C) Fraction of genes with detected unspliced, intronic reads normalized to total number of genes detected in each dataset (total number per sample in legend). Error bars indicate standard error of the mean for 2 replicates. (D) (top) bulk poly(A) tail length density distribution of nuclei from HeLa S3 nuclei control and PlaB experiments for replicate datasets. (bottom) histogram of intronic poly(A) tail length normalized to total read counts per dataset for HeLa S3 nuclei control and PlaB splicing inhibition. Number of reads per dataset/replicate for bulk/intronic are displayed inside legend. (E) Poly(A) tail length distributions for Nanopore direct RNA sequencing. Reads grouped by annotated as intronic, poly(A) site, or post poly(A) site reads for sequencing protocols involving enzymatic poly(A) tailing of RNA or no tailing. Reads were categorized as spliced (top) or unspliced (bottom) by our computational pipeline.

Figure 2

Metabolic labeling indicates rapid shortening of poly(A) tails after synthesis (A) Experimental outline for 4sU pulldown and SLAM-Seq metabolic labeling experiments. (B) Poly(A) tail length distributions for supernatant (SN) and pulldown (PD) fractions after metabolic labeling with 4sU for indicated time points. Dashed distribution indicates poly(A) tail length for intronic reads from HEK metabolic labeling experiments. (C) Poly(A) tail length distributions of all genes, immediate-early genes (IEGs), long noncoding RNAs (lncRNA) and ribosomal protein encoding genes (ribosomal) for different labeling timepoints in 4-SU pulldown experiments.

Figure 3

Biochemical fractionation reveals nuclear deadenylation (A) Experimental outline for biochemical fractionation experiments from HeLa S3 cell lines in cytoplasmic, nucleoplasm and chromatin fractions. FLAM-seq libraries were generated from RNA extracted from fractions. (B) Poly(A) tail length density distributions of cytoplasmic (Cyto), nucleoplasmic (Nuc) and chromatin (Chr) fractions from HeLa S3 cells. Ribbons indicate SD of 12 replicate samples. (C) Poly(A) tail length profiles of intronic reads in HeLa S3 fractions. Ribbons indicate SD of 12 replicate samples. (D) Experimental outline for fractionation of mouse brains into nuclei and cytoplasmic fractions using a Dounce homogenizer. FLAM-seq libraries were generated from RNA extracted from fractions. (E) Poly(A) tail length profiles of mouse brain cytoplasm and nuclei fractions. Bulk distributions include all reads, intronic distributions include unspliced reads. (F) Poly(A) tail length distributions for lncRNAs in HeLa S3 (top) and mouse brain (bottom) fractionation experiments. (G) Comparison of median poly(A) tail length per gene profiles between cellular fractions. (H) Comparison of gene enrichment between nucleus and cytoplasm (inferred from HeLa ENCODE data) and median poly(A) tail length per gene in each fraction.

Figure 4

A mathematical model predicts fast nuclear deadenylation (A) Model description (see STAR Methods). (B) Simulations of mRNA levels in the nucleus (green) and cytosol (red) with the basic model assuming constant export (top left), the model assuming constant export and nuclear decay (ndce model; top right), the model assuming tail-dependent export (nndle model; bottom left), compared to the merged FLAM-seq data from 12 fractionation replicates (bottom right). The average of the estimated parameter distributions was used as input. Cytoplasmic contamination in the nuclear fraction of 0% was assumed. (C) Distributions of cytoplasmic (red) and nuclear (green) deadenylation rates, estimated with models assuming nuclear decay (ndce) or tail-dependent export (nndle), with three contamination values and from 12 fractionation experiments replicates merged (see STAR Methods). (D) Summary sketch: mRNAs with long poly(A) tail are synthesized and rapidly deadenylated in the nucleus. mRNAs with shortened tails are exported to the cytoplasm where they are translated, until progressive deadenylation leaves mRNAs without poly(A) tails, which are decapped and degraded.