Nuclear RNA sequencing of the mouse erythroid cell transcriptome

Area of Science:

• Genomics
• Molecular Biology
• Transcriptomics

Background:

• Mammalian genomes contain a significant portion of transcribed regions beyond protein-coding genes.
• Existing transcriptome studies primarily focus on steady-state messenger RNA (mRNA) levels, overlooking a substantial fraction of the transcribed genome.
• Steady-state mRNA levels are influenced by both transcriptional and post-transcriptional regulation, obscuring a precise understanding of transcriptional output.

Purpose of the Study:

• To compare the nuclear transcriptome with genome-wide RNA polymerase II (RNAPII) occupancy.
• To investigate the correlation between transcriptional output and RNAPII association.
• To identify nuclear-retained long non-coding transcripts and regulatory elements.

Main Methods:

• Deep sequencing of nuclear RNAs (nucRNA-Seq) was performed in parallel with chromatin immunoprecipitation sequencing (ChIP-Seq) for active RNAPII.
• The study utilized mouse anemic spleen erythroid cells.
• Quantification of unspliced transcripts and comparison with steady-state mRNA levels and RNA FISH were conducted.

Main Results:

• Unpliced transcripts quantified by nucRNA-Seq correlate with primary transcript frequencies but differ from steady-state mRNA levels.
• A poor genome-wide correlation was observed between transcriptional output and RNAPII association, particularly due to intergenic regions.
• These intergenic regions are associated with transcription factor binding sites and stable, nuclear-retained long non-coding RNAs.

Conclusions:

• Sequencing the nuclear transcriptome provides a valuable method for assessing the transcriptional landscape of a specific cell type.
• This approach allows for the quantification of unspliced primary transcripts, offering a more direct measure of transcription.
• Nuclear transcriptome sequencing facilitates the identification of nuclear-retained long non-coding RNAs, contributing to a comprehensive understanding of the transcriptome.