To demonstrate that very small RNAs can be used by RNAP as primers for transcription initiation in vivo, Goldman et al.
took advantage of studies investigating the metabolism of small RNAs in bacterial cells. These studies had revealed the existence of a class of enzymes responsible for the degradation of RNAs between 2 and ~5-nt in length 13–17
; such extremely small RNAs have been termed “nanoRNAs” to distinguish them from other classes of small RNAs 16
. In E. coli
the degradation of nanoRNAs is carried out by an essential 3′ to 5′ exonuclease, Oligoribonuclease (Orn) 15
. Thus, inactivation of Orn in E. coli
was shown to result in the accumulation of 2- to ~5-nt nanoRNAs 15
. Accordingly, Goldman et al.
tested whether inactivation of Orn might lead to cellular conditions where a significant fraction of transcription is primed due to the presence of elevated concentrations of 2- to ~5-nt nanoRNAs. Because inactivation of Orn in E. coli
leads to a loss of viability 15
, the authors determined the effect of inactivating Orn in Pseudomonas aeruginosa
, a Gram-negative organism that remains viable in the absence of Orn function 18
Using a specially engineered strain of P. aeruginosa that allows for the artificial depletion of Orn, Goldman et al. first demonstrated that, similar to results obtained in E. coli, loss of Orn function in P. aeruginosa leads to accumulation of 2- to ~4-nt nanoRNAs. Next, to determine whether increasing the intracellular concentrations of 2-to ~4-nt nanoRNAs leads to the widespread use of nanoRNAs as primers for transcription initiation, the authors used high-throughput sequencing to analyze the 5′ ends of primary transcripts on a genome-wide scale.
In vitro studies indicate that 2- to 4-nt RNAs that are complementary to the DNA template can efficiently compete with NTPs for use as primers during transcription initiation provided the 5′ end of the RNA base pairs with promoter sequences between positions −3 and +1 and the 3′ end of the RNA base pairs with promoter sequences between positions +1 and +3 11, 12, 19–23
. (Note that by convention the position where de novo
transcription begins is designated +1.) Thus, use of 2- to 4-nt RNAs to prime transcription initiation in vitro can, in some cases, result in production of transcripts that are extended in length by 1, 2, or 3 nt at the 5′ end compared with transcripts that are produced in reactions performed in the presence of NTPs alone. The authors therefore reasoned that if 2- to 4-nt RNAs could also prime transcription initiation in vivo, increasing the intracellular concentration of 2- to 4-nt nanoRNAs should result in 5′ end alterations for primary transcripts produced from a significant fraction of cellular promoters. Consistent with this prediction, using high-throughput sequencing Goldman et al.
found that depletion of Orn did, in fact, cause the appearance of significant amounts of primary transcripts that were, on average, 1, 2, or 3 nt longer than those observed in non-depleted cells.
The authors presented several lines of evidence supporting the conclusion that the observed changes in the average distributions of the 5′ ends of primary transcripts upon depletion of Orn are a direct consequence of widespread use of 2- to 4-nt nanoRNAs as primers for transcription initiation. Among these, the two most important are the following. First, depleting Orn in the presence of a heterologous nanoRNA-degrading enzyme, the NrnB protein from Bacillus subtilis 17
, did not result in global alterations in the average distributions of the 5′ ends of primary transcripts. This finding established that the observed change in the distribution of the 5′ ends of transcripts that occurs upon depletion of Orn was a direct consequence of the accumulation of 2- to ~4-nt nanoRNAs (and not due to an unrelated consequence of Orn depletion). Second, it is well documented that alterations in the intracellular concentrations of NTPs can lead to changes in the position where transcription initiation begins [reviewed in 24
]. (This phenomenon, which is termed NTP-dependent start point switching, is discussed further below.) Thus, it was important to rule out the possibility that the observed changes in the average distributions of the 5′ ends of primary transcripts upon depletion of Orn were not due simply to any alterations in NTP concentrations. To investigate this possibility, Goldman et al.
compared the average distribution of the 5′ ends of transcripts carrying a 5′ triphosphate group, i.e. those potentially derived from a de novo
initiation event, with the average distribution of the 5′ ends of all transcripts regardless of the phosphorylation status of the 5′ end. Strikingly, this comparison revealed that whereas depletion of Orn results in a pronounced alteration in the distribution of the 5′ ends of transcripts when all 5′ ends are considered, depletion of Orn results in only a modest change in the average distribution of transcripts carrying a 5′ triphosphate group. These data rule out the possibility that the pronounced changes in the average distributions of the 5′ ends of transcripts are a consequence of alterations in the abundance of NTPs that, in turn, alters the position of de novo
initiation. Consistent with this assertion, the authors presented evidence that the intracellular concentrations of NTPs were identical in Orn-depleted cells and cells in which Orn was not depleted.