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Quartet of New Papers Provide New Insights in Transcription

NEW YORK (GenomeWeb News) – RNA polymerase II can produce sense and antisense transcripts both upstream and downstream of transcription start sites, generating small RNAs that may themselves participate in transcriptional regulation, according to four new papers appearing online today in Science Express.
In the first of these papers, Massachusetts Institute of Technology biologist Philip Sharp led a team of researchers who found evidence for DNA transcription occurring in both directions from some transcription start sites in several cell types and described a new class of small RNAs that they called transcription start site-associated RNAs or TSSa-RNAs.
“People have been studying transcription for a long time and [have] never seen this kind of transcription before,” lead author Amy Seila, a post-doctoral researcher in Sharp’s lab, said in a statement. “It looks like we have a polymerase that binds as we thought, but we also see a polymerase that appears to be pointing in the wrong direction, going upstream from the transcription start site.”
By analyzing more than eight million short RNA sequence reads from cDNA libraries representing both mouse embryonic stem cells and differentiated cells, the team detected 16 to 30 nucleotide TSSa-RNAs associated with more than half of the genes examined.
“Divergent transcription is likely a common feature of mammalian [transcription start sites] given the presence of TSSa-RNAs in all cell types examined in this study,” Sharp and his team noted.
In particular, genes that were more highly expressed in mouse embryonic stem cells also tended to have more TSSa-RNAs associated with them. In addition, about 80 percent of the TSSa-RNAs were detected at promoters with a high frequency of cytosine and guanine nucleotides.
TSSa-RNAs also tended to associate with promoters enriched for bound RNA polymerase II and those with histone H3 lysine 4 trimethylated chromatin. But only downstream transcripts were associated with H3K79-dimethylated histones, a marker of RNA polymerase II elongation, suggesting elongation does not occur upstream.
“These results suggest that divergent transcription over short distances is common for active promoters and may help promoter regions maintain a state poised for subsequent regulation,” the authors noted.
Meanwhile, another team of researchers led by Cornell University molecular biologist and geneticist John Lis used a new method called Global Run-On-Sequencing, or GRO-seq — generating nuclear run-on RNAs and subjecting them to large-scale parallel sequencing using the Illumina 1G sequencing platform — to map the “position, amount, and orientation of transcriptionally-engaged RNA polymerases genome-wide.” Their results suggest divergent RNA polymerase II transcription and paused transcription are relatively common.
“[M]ost promoters have an engaged polymerase upstream and in an orientation opposite to the annotated gene,” the authors wrote. “This divergent polymerase is associated with active genes, but does not elongate effectively beyond the promoter.”
Using their sequencing-based approach, the researchers detected antisense transcripts for nearly 59 percent of the genes in human lung fibroblast nuclei, most mapping near transcription start sites.
They also detected RNA polymerase II activity both upstream and downstream of transcription start sites. “A prominent and surprising feature of the GRO-seq profiles around transcription start sites is the robust signal from an upstream, divergent, engaged polymerase,” Lis and his colleagues wrote. 
Again, divergent transcription tended to occur on genes with active promoters, promoters with high cytosine and guanine levels, and higher overall transcription levels, though upstream polymerases didn’t usually exhibit productive elongation.
In a third paper, a Danish research team used RNA tiling microarray analysis to look at the RNA transcripts present in cells lacking cellular components involved in RNA breakdown. They detected short, unstable, polyadenylated RNAs, which they call promoter upstream transcripts or PROMPTs, generated in both the sense and antisense direction, 500 to 2,500 bases upstream of active transcription start sites.
Senior author Torben Heick Jensen, a researcher at Denmark’s Aarhus University, and his team used ENCODE tiling arrays and quantitative RT-qPCR to look at the RNAs present in normal HeLa cells and HeLa cells lacking components of the exonucleolytic RNA exosome.
Based on the RNAs they discovered and subsequent rapid amplification of cDNA ends experiments, the researchers concluded that RNA polymerase II can function in either orientation upstream of gene promoters. Their experiments indicate that promoter upstream transcripts are associated with active transcription, RNA polymerase II activity, acetylated histones, and gene activity.
Jensen and his colleagues speculated that PROMPTs may participate in transcriptional regulation by influencing promoter methylation. They also suggested that these transcripts may occur whenever open chromatin is present.
“Clearly, the generality of the PROMPT phenomenon hints at a more complex regulatory chromatin structure around the [transcription start site] than was previously anticipated,” Jensen and his colleagues noted.
Finally, researchers from Johns Hopkins University found evidence for widespread antisense transcription in human cells. After developing a technique for quantifying the transcripts derived from each DNA strand the researchers assessed the “antisense transcriptomes” for five different types of human cells. They detected non-randomly distributed antisense transcripts for between 2,900 and 6,500 human genes.
“Antisense transcripts ... appear to be a pervasive feature of human cells, suggesting that they are a fundamental component of gene regulation,” senior author Kenneth Kinzler and his colleagues wrote.
Using an approach called asymmetric strand-specific analysis of gene expression or ASSAGE, which relies on sequencing complementary DNA fragments generated from bisulfite treated RNA, the researchers pinned down the DNA strand of origin for millions of RNA transcripts.
When they examined genes with five or more distinct tags, Kinzler and his team found that almost 82 percent of genes generated mainly sense transcripts, 2.5 percent generated mainly antisense transcripts, and nearly 16 percent generated both. They detected another 6,457 genes with at least two antisense tags, with antisense tags generally clustering near transcription initiation sites.
The team found similar patterns in four other types of human cells, though the genes predominantly expressing each type of transcripts varied by cell type leading the researchers to speculate that antisense tag expression is cell or tissue-specific.
Together, the studies suggest that the transcription machinery is more complex than previously imagined. Researchers speculated that the newly described divergent and antisense aspects of transcription may have regulatory roles. But, they noted, more research is necessary to clarify the transcriptional landscape in cells.
“This is only the beginning of the story,” MIT’s Sharp said in a statement.
Sharp shared a Nobel Prize with New England Biolabs researcher Richard Roberts in 1993, for his work on introns and messenger RNA splicing.

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