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| Status |
Public on Oct 27, 2020 |
| Title |
Widespread transcriptional readthrough caused by Nab2 depletion leads to chimeric transcripts with retained introns |
| Organism |
Saccharomyces cerevisiae |
| Experiment type |
Other
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| Summary |
Nascent RNA sequencing has recently revealed that pre-mRNA splicing can occur shortly after the intron emerges from RNA polymerase II (Pol II). Differences in co-transcriptional splicing profiles suggest regulation by cis- and/or trans-acting factors. Here we used Single Molecule Intron Tracking (SMIT) in budding yeast to identify a cohort of regulators by machine learning. One candidate, Nab2, displayed reduced co-transcriptional splicing of some pre-mRNAs when depleted. Unexpectedly, these splicing defects were attributable to readthrough transcription, which was revealed by long read sequencing of nascent RNA; individual readthrough transcripts induced by Nab2 depletion sometimes spanned multiple genes. Thus, Nab2 regulation of splicing was indirect. Moreover, unspliced transcripts displayed downstream readthrough in both control and Nab2-depleted cells, highlighting the coupling between splicing and 3′ end formation. We conclude that Nab2 is required for proper 3′ end processing, which ensures both transcription termination as well as proper splicing of downstream genes.
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| Overall design |
Single Molecule Intron Tracking (SMIT) was performed for 2 replicates each of 6 different yeast deletion strains as well as the wild type control. Additional SMIT libraries were prepared for Anchor-Away strains (Nab2-AA and Control-AA). Finally, long read sequencing libraries were prepared from nascent RNA extracted from the Nab2-AA and Control-AA samples in duplicate.
Please note that the 'Nab2AA_ControlAA_fast5.tar.gz' (linked to the Long read Nab2-AA rep1 sample records) contains raw data for all four "Long read" samples. As the algorithm for converting and demultiplexing the fast5 into fastq files is proprietary software and not all users will have access to the exact form/version used, both fastq and fast5 data were made available as SRA and GEO records, respectively.
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| Contributor(s) |
Alpert T, Straube K, Carrillo Oesterreich F, Neugebauer KM |
| Citation(s) |
33113357 |
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| Submission date |
Aug 12, 2020 |
| Last update date |
Jan 04, 2021 |
| Contact name |
Tara Diane Alpert |
| E-mail(s) |
tara.alpert@yale.edu
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| Organization name |
Yale University
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| Department |
Molecular Biophysics and Biochemistry
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| Lab |
Karla Neugebauer
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| Street address |
333 Cedar St.
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| City |
New Haven |
| State/province |
CT |
| ZIP/Postal code |
06511 |
| Country |
USA |
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| Platforms (2) |
| GPL17342 |
Illumina HiSeq 2500 (Saccharomyces cerevisiae) |
| GPL25739 |
MinION (Saccharomyces cerevisiae) |
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| Samples (24)
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| Relations |
| BioProject |
PRJNA656824 |
| SRA |
SRP277240 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE156133_RAW.tar |
193.8 Gb |
(http)(custom) |
TAR (of BED, TAR, TXT) |
| GSE156133_dSMIT_deletions_rep1.txt.gz |
3.2 Kb |
(ftp)(http) |
TXT |
| GSE156133_dSMIT_deletions_rep2.txt.gz |
3.3 Kb |
(ftp)(http) |
TXT |
| GSE156133_longread_fractionspliced.txt.gz |
4.7 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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