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Mar 15, 2012 (Vol. 32, No. 6)

RNA Advances Reshape Prevailing Wisdom

  • microRNA

    Approximately 98% of the transcriptional output of the human genome is represented by noncoding RNAs, which play fundamental roles in shaping the complexity of genome architecture and dynamics. MicroRNAs, a class of small noncoding RNAs that post-transcriptionally regulate gene expression, have important roles in processes that govern development, physiology, and disease. Two-thirds of the human microRNAs are located within introns of protein-encoding genes.

    “For these genes, there is a unique possibility that microprocessing and splicing is coupled, either directly through protein-protein interactions or indirectly through the pre-mRNA where both macromolecular complexes assemble,” says Carl D. Novina, M.D., Ph.D., associate professor of microbiology and immunobiology at the Dana-Farber Cancer Institute and Harvard Medical School.

    In a study that analyzed miR-211, a microRNA that suppresses melanoma invasion and is expressed from intron 6 of melastatin, Dr. Novina and colleagues recently showed that miR-211 microprocessing promotes splicing of exons 6 and 7, and mutations at the 5 splice site reduced miR-211 biogenesis. These findings revealed a novel physical and functional coupling between gene splicing and microRNA biogenesis and unveiled a new layer of regulation.

    “In this feed-forward model, microprocessing facilitates splicing and splicing facilitates microprocessing at intronic microRNA loci. It is possible that microRNAs may play a broader role in regulating gene expression at the level of mRNA maturation in addition to their roles in regulating mRNA stability and translation,” explains Dr. Novina.

  • Click Image To Enlarge +
    Researchers at the University of Miami School of Medicine are using RNA-Seq to compare isoforms expressed in peripheral neurons from dorsal root ganglia, which are able to regenerate after injury, with those from cerebellar granule neurons, which do not regenerate.

    “Performing RNA sequencing works fantastically well, and it does not cost very much, but the software for analyzing the giant datasets is very challenging and still evolving, making the informatics side really hard,” says Vance Lemmon, Ph.D., professor of neurological surgery at the University of Miami School of Medicine.

    Dr. Lemmon and colleagues recently used RNA-Seq to compare isoforms expressed in peripheral neurons from dorsal root ganglia, which are able to regenerate after injury, with those from cerebellar granule neurons, which do not regenerate. This comparative approach unveiled over 8,000 differentially expressed isoforms between the two cell types.

    “After comparing gene expression in different types of neurons, we hope to exploit this information and transfer it from neurons that regenerate to neurons that do not regenerate, to identify targets that promote neuronal regeneration in the central nervous system,” explains Dr. Lemmon.

    In addition to being very economical, RNA-Seq has several additional advantages. “This approach offers the additional opportunity to obtain information about isoforms that have never been studied and might not be in any databases,” says Dr. Lemmon. The possibility to use very small amounts of starting material also helps gain insight into the biology of defined groups of cells.

    “With the possibility to identify all the RNA species from such a small amount of starting material, we can define much more precisely the specific roles they play in the different cell types from different brain regions, or under many different conditions, such as during development, disease, or injury, and this represents more information than anyone was able to get in biology before, and it is all happening in real time,” says Jessica K. Lerch, Ph.D., first author of the study.

  • Gene Expression

    Click Image To Enlarge +
    Comparison of RNA-seq to microarray or subtractive hybridization approaches to study differential gene expression. When gene-expression levels were compared between sensory neurons and cerebellar neurons many more genes were found by RNA-seq. In addition, several thousand unique isoforms were found that could not be found with the older methods. [Adapted from Lerch et al., PLoS One. (2012) 7:e30417.]

    For a long time, the prevailing view about regulation of gene expression was that after the transcription initiation was finished, the subsequent process of elongation was not intensively regulated or controlled. “This view changed once several laboratories, including ours, had shown the existence of factors that control the rate and processivity of transcriptional elongation catalyzed by RNA polymerase II,” says Ali Shilatifard, Ph.D., investigator at the Stowers Institute for Medical Research.

    An important finding that emerged relatively recently was that certain mutations in genes that regulate this process are linked to human malignancies and other diseases, elevating transcriptional elongation control to the center stage of cellular processes relevant for human development and disease pathogenesis.

    “In the past eight to ten years, we have learned not only that transcriptional elongation control is central to the process of gene expression, but also that there is actually a gene class specificity such that an elongation factor could exist for every class of gene,” explains Dr. Shilatifard.

    RNA biology has shaped some of the most significant moments in the history of life sciences. From the 1967 discovery that RNA can perform catalytic functions, to the seminal finding in the early 1970s that it can be copied into DNA by reverse transcriptase, and culminating with the more recent years in which considerable focus has centered on splicing and RNA interference, RNA biology has become one of the most dynamic research areas.

  • Moving RNA Devices into the Clinic

    Several characteristics of RNA, which include its ability to function as a sensor and a regulator, and the ease to design it into various structures, facilitated the emergence of synthetic RNA devices that can be exploited to modulate cellular activities.

    “There are multiple key ways in which RNA devices will be important in the clinic,” says Christina D. Smolke, Ph.D., assistant professor of bioengineering at Stanford University.

    In addition to the lack of a requirement for heterologous proteins, which can often elicit nonspecific immunogenic responses in human cells, RNA devices are encoded within very compact platforms that do not place a large burden on the cells. One of their more immediate therapeutic applications is within cell-based therapies, such as stem cell therapy or immunotherapy.

    While T-cells or stem cells used in cell-based therapies have to persist in the organism long enough to exert their specific functions, their unchecked proliferation could contribute to a different type of cancer and represents a major concern. RNA devices address this shortcoming by providing a programmable control system that precisely regulates therapeutic activities in response to different inputs.

    “These inputs may be an approved, nontoxic drug molecule, or an endogenous disease signal that activates cells when they are in the specific disease microenvironment,” Dr. Smolke adds. By building a synthetic RNA device that includes an aptamer as the modular sensor and a hammerhead ribozyme as the gene-regulatory component, Dr. Smolke and colleagues recently illustrated the possibility to trigger the in vivo proliferation of T cells and modulate their growth rate in response to drug molecules.

    “The cells will be engineered ex vivo to harbor genetic circuits containing RNA control devices and then ultimately introduced back into the patients,” says Dr. Smolke. The engineering of specific therapeutic activities into T cells enables investigators to regulate their proliferation and activation in the human body, allowing better temporal and spatial control and providing a safer and more effective therapy. The modularity enables the device to be easily tailored to specific therapeutic needs and represents an additional advantage.

    “The proof of concept is there, and the next steps are developing integrated, robust systems, directing them toward the very relevant clinical activities that have to be regulated, and moving them into systemic animal models.”

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