Genome-wide quantification of RNA flow across subcellular compartments reveals determinants of the mammalian transcript life cycle

Abstract

Dissecting the myriad regulatory mechanisms controlling eukaryotic transcripts from production to degradation requires quantitative measurements of mRNA flow across the cell. We developed subcellular TimeLapse-seq to measure the rates at which RNAs are released from chromatin, exported from the nucleus, loaded onto polysomes, and degraded within the nucleus and cytoplasm. These rates varied substantially, yet transcripts from genes with related functions or targeted by the same transcription factors and RNA binding proteins flowed across subcellular compartments with similar kinetics. Verifying these associations uncovered roles for DDX3X and PABPC4 in nuclear export. For hundreds of genes, most transcripts were degraded within the nucleus, while the remaining molecules were exported and persisted with stable lifespans. Transcripts residing on chromatin for longer had extended poly(A) tails, whereas the reverse was observed for cytoplasmic mRNAs. Finally, a machine learning model identified additional molecular features that underlie the diverse life cycles of mammalian mRNAs.

Pre-mRNA splicing order is predetermined and maintains splicing fidelity across multi-intronic transcripts

“…Two biological replicates from K562 chromatin-associated RNA were sequenced. We also analyzed two of chromatin RNA replicates as well as two cytoplasmic and two total RNA samples (Supplemental Table 1) that were produced in 58 . For all downstream analyses in K562 cells, replicates 3 and 4 produced in this study were combined into replicate A and replicates 1 and 2 produced in 58 were combined into replicate B (Supplemental Table 1) to increase coverage.…”

“…We also analyzed two of chromatin RNA replicates as well as two cytoplasmic and two total RNA samples (Supplemental Table 1) that were produced in 58 . For all downstream analyses in K562 cells, replicates 3 and 4 produced in this study were combined into replicate A and replicates 1 and 2 produced in 58 were combined into replicate B (Supplemental Table 1) to increase coverage. For analysis of sMN differentiation, we sequenced biological duplicates for days 9 and 14, while we were limited to sequencing a single biological replicate for day 4 due to lower cell counts at this earlier timepoint.…”

The transcript interactome of skeletal muscle RNA binding protein motif 3 (RBM3)

“…Lastly, RBM3 was enriched with transcripts related to ribonucleoprotein granule colocalization, including Ddx1 and Hnrnpa2b1; thus, RBM3 may regulate the abundance of, and be found complexed with machinery necessary for protein synthesis (Dresios et al, 2005 ). Indeed, it has been suggested that a large percentage of mRNA is degraded by decay pathways in the nucleus (~40%), and that a critical determinant to ensuring nuclear to cytoplasmic transport involves specific mRNA sequences for RBP binding (Houseley & Tollervey, 2009 ; Smalec et al, 2022 ). Therefore, RBPs such as RBM3 may ensure delivery of mRNAs to cytosolic ribosomes for translation, and context‐dependent degradation of transcripts in situations of cellular stress.…”

Subcytoplasmic location of translation controls protein output

“…Instead, these mRNAs had the lowest transcription rates which suggests that these mRNAs are either produced at a low rate or have high cotranscriptional degradation rates (Fig. 2C, 2D) (Smalec et al, 2022).…”

“…Instead, these mRNAs had the lowest transcription rates which suggests that these mRNAs are either produced at a low rate or have high cotranscriptional degradation rates (Fig. 2C, 2D) (Smalec et al, 2022). CRER-enriched mRNAs had higher production and stability values and encode proteins with the highest expression levels (Fig.…”