Thomas R Gingeras

Thomas R. Gingeras is at the Department of Functional Genomics, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 11724, USA.
e-mail: tom_gingeras@affymetrix.com

Figure 1 : Three levels of resolution showing pervasive interleaved transcription.
The clustering of genes in the upper portion of the figure cloaks the overlapping transcription of protein-coding and noncoding transcripts observed within and between genic regions, as depicted in the middle panel. These transcripts also point to multiple regulatory regions (triangles and circles) that are positioned within genes and present on opposite strands. The ultimate fate of some of these long transcripts (for example, promoter-associated long RNAs; PALRs) is to provide short RNAs such as microRNAs, promoter-associated short RNAs (PASRs) and termini-associated short RNAs (TASRs).
(Gene Clusters: Purple), (Transcription Start Sites (TSS): Green.), (Primary Transcripts (PT): Blue).

Consistent with data obtained from studies of human 5, mouse 6 and fly 7, the authors find that virtually all of the euchromatic genome is transcribed. However, two things of note distinguish this observation from other genome-wide transcription studies. The first involves the technical approach used by the authors to obtain their transcriptome maps. Specifically, the authors used an antibody (S9.6) raised against RNA-DNA duplexes to identify duplex regions formed on DNA probes that were part of whole-genome tiling arrays. This allowed for the strand-specific designation of each of the detected transcribed regions without the use of reverse transcriptase or RNA-DNA amplification techniques, which can have issues associated with the production of only single-stranded products. The results using this technical approach are notable in both their range of detection (7,600-fold) and their specificity (reduction in hybridization signal of 80- and 20,000-fold with one and two base mismatches, respectively). However, given that these results are dependent upon the use of an immunological approach, there is a concern as to whether these results will be reproducibly achieved by other laboratories using even slightly different labeling protocols, antibody preparations from the hybridoma cell line or detection array systems. Similar concerns have plagued chromatin immunoprecipitation studies. Nevertheless, the results achieved using this approach provide a fresh alternative to achieve strand-specific RNA maps using tiling arrays.

The second notable aspect of this study focuses on the characterization of previously unannotated transcribed genomic regions. One class of unannotated transcription in S. pombe noted by Dutrow et al. involves the detection of widespread antisense transcription. The authors indicate that the detection of antisense transcription in S. pombe was less prevalent when polyadenylated (poly(A)+) RNA was mapped compared to total RNA. The authors interpret this to be consistent with the possibility of the antisense transcripts being poly(A)- or possessing reduced length polyadenylation. Dutrow et al. suggest that the cause of this antisense transcription is opportunistic on the basis of the negative correlation with histone H3 occupancy and the positive correlation of coordinated expression of sense-antisense transcription during conditions of gene expression changes (for example, heat shock) and the presence of histone H3 lysine 36 trimethylation (H3K36me3). However, the conclusion that S. pombe antisense transcription is in large measure composed of poly(A)- RNA implies that such transcripts would likely have a rapid turnover rate and be relatively short-lived. If correct, this observation would represent a significant difference compared to that observed in mouse and human cells 3, 8, in which most of the detected antisense transcription is detected in poly(A)+ RNA samples and contains reasonable length polyadenylation, as revealed by cDNA sequencing. Direct empirical determination of the polyadenylation state of most antisense transcripts in S. pombe would be straightforward and would not only confirm these observations but also provide possible insights as to why such a marked contrast is observed between fission yeast and higher eukaryotic cells.

Opportunistic or deterministic?

Another characteristic of antisense transcripts observed by Dutrow et al. involves the previously uncharacterized transcribed regions flanking the tRNAs residing in the imr repeats found in the heterochromatic centromeric regions. The regions flanking the tRNA genes seem to be transcribed on the antisense strand relative to the tRNA gene. Such flanking antisense transcription is reported not to be observed at tRNA genes found in euchromatin regions. Again, the authors see this antisense transcription as the result of opportunistic conditions set up by the directed transcription of the tRNA genes. Extending this line of thought, the authors conclude that the genome-wide baseline transcription observed along intergenic regions is also opportunistic and indicative of the chromatin state of these regions.

Although initially attractive, this explanation places the role of transcription of a large portion of a genome as a passive and baseline condition in the cell. Such a promiscuous role for transcription is troubling for two reasons. First, as indicated by the authors themselves, the RNA detected in their studies reflects a steady state condition in the cells, and thus, these molecules are not likely to be short-lived. This is especially the case when the same transcribed regions are observed at multiple time points during development or in response to external stimuli, as seen in this study. These nontransient RNAs within cells are thus likely to be immediately associated with a diverse collection of RNA-binding proteins. The roles of these proteins are substantial but their abundance in a cell is not. An organizational strategy that uses opportunism on such a global scale greatly increases the requirement for regulatory complexity to discern the products of opportunism from determinism so as to judiciously use the limited RNA binding–protein resources of the cell and sets up conditions for creating transcripts that have the same regulatory signals as transcripts created in a deterministic fashion.

One of the results from the pilot ENCODE studies may point to a less opportunistic reason for the synthesis of such transcripts 1. An analysis of ENCODE regions aligned for 23 mammals and 5 other vertebrates showed significant enrichment for short islands of conservation within transcripts of unknown function (TUFs), despite absence of detectable conservation when the enrichment was averaged across the whole length of each transcribed region. Thus, long intergenic or antisense transcripts can be made in a directed and regulated fashion in order to provide short functional RNAs (for example, microRNA primary transcripts) or allow for the rapid evolution of sense-antisense transcript pairs (Fig. 1). It seems likely that additional studies will be undertaken involving transcripts originating from unannotated regions to determine whether stable short RNAs are also found mapping to these same regions and which are enriched in evolutionary conserved sequences, as has been observed in human cell lines 3.

NetworkEditor's Perspective: Euchromatin is pervasively active for gene transcription.

This new technique of Natalie Dutrow , David Nix , Derick Holt , Brett Milash , Brian Dalley , Erick Westbroek , Timothy Parnell, and Bradley Cairns is designed for analyzing intact DNA-RNA hybrids at sites of active gene transcription, and offers both markedly higher resolution and the ability to distinguish each DNA strand at a particular gene locus. Most if not all of the DNA sequences of extended euchromatin appear to be undergoing active transcription to RNA. Protein-coding RNAs, non-coding RNAs, transfer RNAs, microRNAs, and both transcription start sites and and RNA splicing sites can be detected.  Some gene loci appear to produce paired sense-antisense RNA doublets, and on occasion these paired RNA-RNA  doublets involve coding RNAs and/or transfer RNAs. Telomere sites, centromere sites, and heterochromatin-transition sites can also be analyzed. This work has been done on yeast species, and is now being extended to more complex species.
 

1. Schwartz JC, Younger ST, Nguyen N-B, Hardy DB, Monia BP, Corey DR, and Janowski BA,
"Antisense transcripts are targets for activating small RNAs".

2. Frenster JH, and Hovsepian JA, "Models of  Embryonic RNA Initiating and Reverting Adult Neoplasms".

3. Ogawa Y, Sun BK, and Lee JT,
"Intersection of the RNA Interference and X-Inactivation Pathways".

4. Place RF, Li L-C, Pookot D, Noonan EJ, and Dahiya R, "MicroRNA-373 induces expression of genes with complementary promoter sequences".

5. Borel C, Gagnebin M, Gehrig C, Kriventseva EV, Zdobnov EM, and Antonarakis SE,
"Mapping of Small RNAs in the Human ENCODE Regions".

6. Zhu X, Ling J, Zhang L, Pi W, Wu M, and Tuan D, "A facilitated tracking and transcription mechanism of long-range enhancer function".

7. Frenster JH, and Hovsepian JA, "DNase-I Ultrastructural Probe Sites and Kissing Chromosomes".

8. Han J, Kim D, and Morris KV, "Promoter-associated RNA is required for RNA-directed transcriptional gene silencing in human cells".

Links to Euchromatin Activator RNA Reviews:
Links to Euchromatin Activator RNA Research:
Links to Ultrastructural Probes of DNase I-Sensitive Sites:
Links to RNA as a Therapeutic Agent:
Links to Hodgkin Lymphoma Immuno-Pathology:
Links to Activated T-Lymphocyte Immunotherapy:
Links to Medical Systems Biology:
Links to Selective Gene Transcription:
Links to RNA-Induced Epigenetics:
Links to RNA-Induced Embryogenesis:
Links to RNA and Biological Causality:
Links to Reprogramming and Neoplasia:

A Brief History of Activator RNA:

"Ultrastructural Probes of Active DNA Sites, and the RNA Activators of DNA". (PowerPoint Presentation).