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Tiny strands of genetic material called RNA – a chemical cousin of DNA – are emerging as major players in gene regulation, the process inside cells that drives all biology and that scientists seek to control in order to fight disease.

The idea that RNA (ribonucleic acid) is involved in activating and inhibiting genes is relatively new, and it has been unclear how RNA strands might regulate the process.

RNA experts at UT Southwestern Medical Center found that, contrary to established theories, RNA can interact with a non-gene region of DNA called a promoter region, a sequence of DNA occurring spatially in front of an actual gene. This promoter must be activated before a gene can be turned on.

"Our findings about the underlying mechanisms of RNA-activated gene expression reveal a new and unexpected target for potential drug development," said Dr. David Corey, professor of pharmacology and biochemistry at UT Southwestern and one of the senior authors of the study.

Genes are segments of DNA housed in the nucleus of every cell, and they carry instructions for making proteins. Faulty or mutated genes lead to malfunctioning, missing or overabundant proteins, and any of those conditions can result in disease. Scientists seek to understand the mechanisms by which genes are activated, or expressed, and turned off in order to get a clearer picture of basic cell biology and also to develop medical therapies that affect gene expression.

In previous studies, Dr. Corey and Dr. Bethany Janowski, assistant professor of pharmacology at UT Southwestern and a senior author of the current study, have shown that tiny strands of RNA can be used to activate certain genes in cultured cancer cells. Using strands of RNA that they manufactured in the lab, the researchers showed that the strands regulate gene expression by somehow perturbing a delicate mixture of proteins that surround DNA and control whether or not genes are activated.

Until now, however, it was not clear exactly how the synthetic RNA strands affected that mix of regulating proteins.

In the current study, also carried out in cancer cell cultures, the UT Southwestern research team discovered an unexpected target for the manufactured RNA. The RNA did not home in on the gene itself, but rather on another type of RNA produced by the cell, a so-called noncoding RNA transcript. This type of RNA is found in association with the promoter regions that occur in front of the gene. Promoter regions, when activated, act essentially as a "start" command for turning on genes.

The researchers found that their man-made RNA strand bound to the RNA transcript, which then recruited certain proteins to form an RNA-protein complex. The whole complex then bound to the promoter region, an action that could then either activate or inhibit gene expression.

"Involvement of RNA at a gene promoter is a new concept, potentially a big new concept," Dr. Janowski said. "Interactions at gene promoters are critical for understanding disease, and our results bring a new dimension to understanding how genes can be regulated."

Until recently, many scientists believed that proteins alone control gene expression at promoters, but Drs. Corey and Janowski's results suggest that this assumption is not necessarily true.

"By demonstrating how small RNAs can be used to recruit proteins to gene promoters, we have provided further evidence that this phenomenon should be in the mainstream of science," Dr. Corey said.

Although using synthetic RNA to regulate gene expression and possibly treat disease in humans is still in the future, Dr. Corey noted that the type of man-made RNA molecules employed by the UT Southwestern team are already being used in human clinical trials, so progress toward the development of gene-regulating drugs could move quickly.

Note for Ribonucleic Acid
Ribonucleic acid (RNA) is a nucleic acid and consists of a long chain of nucleotide units. Each nucleotide consists of a nitrogenous base, a ribose sugar, and a phosphate. RNA is very similar to DNA, but differs in a few important structural details: in the cell RNA is usually single stranded, while DNA is usually double stranded. RNA nucleotides contain ribose while DNA contains deoxyribose (a type of ribose that lacks one oxygen atom), and RNA has the nucleotide uracil rather than thymine which is present in DNA.

RNA is transcribed from DNA by enzymes called RNA polymerases and is generally further processed by other enzymes. RNA is central to the synthesis of proteins. Here, a type of RNA called messenger RNA carries information from DNA to structures called ribosomes. These ribosomes are made from proteins and ribosomal RNAs, which come together to form a molecular machine that can read messenger RNAs and translate the information they carry into proteins. There are also many RNAs involved in modifying other RNAs; some of it causing maturation of RNAs, other resulting in altered expression or products of genes.

Synthesis of RNA is usually catalyzed by an enzyme—RNA polymerase—using DNA as a template, a process known as transcription. Initiation of transcription begins with the binding of the enzyme to a promoter sequence in the DNA (usually found "upstream" of a gene). The DNA double helix is unwound by the helicase activity of the enzyme. The enzyme then progresses along the template strand in the 3’ to 5’ direction, synthesizing a complementary RNA molecule with elongation occurring in the 5’ to 3’ direction. The DNA sequence also dictates where termination of RNA synthesis will occur.

RNAs are often modified by enzymes after transcription. For example, a poly(A) tail and a 5' cap are added to eukaryotic pre-mRNA.

There are also a number of RNA-dependent RNA polymerases as well that use RNA as their template for synthesis of a new strand of RNA. For instance, a number of RNA viruses (such as poliovirus) use this type of enzyme to replicate their genetic material. Also, it is known that RNA-dependent RNA polymerases are required for the RNA interference pathway in many organisms.

Note for non-coding RNA
A non-coding RNA (ncRNA) is any RNA molecule that is not translated into a protein. A previously used synonym, particularly with bacteria, was small RNA (sRNA). However, some ncRNAs are very large (e.g. Xist). Less-frequently used synonyms are non-messenger RNA (nmRNA), small non-messenger RNA (snmRNA), or functional RNA (fRNA). The DNA sequence from which a non-coding RNA is transcribed as the end product is often called an RNA gene or non-coding RNA gene.

Non-coding RNA genes include transfer RNA (tRNA) and ribosomal RNA (rRNA), small RNAs such as snoRNAs, microRNAs, siRNAs and piRNAs and lastly long ncRNAs that include examples such as Xist, Evf, Air, CTN and PINK. The number of ncRNAs encoded within the genome is unknown, however recent transcriptomic and microarray studies suggest the existence of over 30,000 long ncRNAs and at least as many small regulatory RNAs within the mouse genome alone. Since most of the newly identified ncRNAs have not been validated for their function, it is possible that the majority of them are meaningless (e.g. non-functional or truncated transcript).

It was formerly believed that the main role for RNA was to code for protein, though there were the recognized exceptions of mRNA (messenger), and tRNA (transfer). It was assumed that any leftover ncRNA which served none of those roles must usually be mere "junk" coding. Some might conceivably be coopted later by chance in the course of evolution, but it was otherwise assumed to be useless.

The term ncRNA has been used, in addition to its above definition, to describe regions of mRNA that are functional at the RNA level, i.e. they have a biological function other than coding for protein even though they are on a protein-coding mRNA, for example riboswitches and the SECIS element. They may even overlap with protein-coding sequence and are thus dual-functional: at the RNA level and at the protein level (e.g. SgrS RNA and RNAIII). However, these conflict with the Sequence Ontology's definition of ncRNA, which requires that a RNA does not contain any protein-coding sequence in order to be labeled ncRNA.

Several publications have started using the term functional RNA (fRNA), as opposed to ncRNA, to describe regions functional at the RNA level that may or may not be stand-alone RNA transcripts. Therefore, every ncRNA is a fRNA, but there exist fRNA (such as riboswitches, SECIS elements, and other cis-regulatory regions) that are not ncRNA. Yet the term fRNA could also include mRNA as this is RNA coding for protein and hence is functional. Additionally artificially evolved RNAs also fall under the fRNA umbrella term. Some publications state that the terms ncRNA and fRNA are nearly synonymous.

Other researchers from UT Southwestern involved in the research were lead author and student research assistant Jacob Schwartz; student research assistant Scott Younger; and research associate Ngoc-Bich Nguyen. Researchers from the University of Western Ontario and ISIS Pharmaceuticals also participated.

The research was supported by the National Institutes of Health and the Welch Foundation.

In figure, Drs. David Corey and Bethany Janowski's research into the underlying mechanisms of ribonucleic acid-activated gene expression has revealed a new and unexpected target for potential drug development.