|
The discovery of small non-coding RNAs capable of repressing, silencing, or even enhancing gene expression continues to inspire researchers who study the regulation of gene expression. One of those researchers, Dr. Victor Ambros, Professor of Genetics, Dartmouth Medical School, is also one of the first scientists to discover microRNAs—a class of small non-coding RNAs that regulate gene expression. Recently, Dr. Ambros’s lab has begun to apply TaqMan®-based real-time PCR assays from Applied Biosystems to his lab’s investigations of how microRNAs influence the fate of cells in flies, worms, and more recently in human brain cancer tumors.
Through the use of real-time PCR assays, which are capable of detecting and quantifying mature microRNA (miRNA) levels in different cell types, Dr. Ambros hopes to explain how miRNAs regulate the expression of genes involved in timing pathways as cells in worms and flies differentiate into different cell types during development. Also, in a collaboration with Dr. Mark Israel, Director of the Norris Cotton Cancer Center, he has been using real-time PCR assays to assess different patterns of miRNA expression as a potential means for discerning different tumor types in cancerous human brain tissue samples.
Discovery of MicroRNAs
In 1993, Dr. Victor Ambros, Rosalind Lee, and Dr. Rhonda Feinbaum published a finding about molecular events that transpire in the development of Caenorhabditis elegans. In that same paper, they also described for the first time the function of a small non-coding RNA gene named lin-4 (lineage-abnormal-4). At the time, they did not realize the significance of this finding (1).
“We discovered that lin-4 encoded a small RNA, but it was the only known case of that kind of small RNA for six or seven years, so, at the time, it wasn’t thought of as being a discovery pertaining to wider phenomona,” recalls Dr. Ambros. “It was considered as something interesting, and people wondered if there were other smaller RNAs like it.”
Lin-4, a gene encoding a 22-nucleotide (nt) non-coding RNA molecule that represses translation of mRNAs into proteins involved in the timing and sequence of postembryonic development in C. elegans, was the first of a class of regulatory genes that have come be known as miRNAs. Since this discovery, miRNAs have been found scattered throughout the genomes of almost all multicellular organisms, and play a significant role in regulating gene expression.
“MicroRNAs are regulatory molecules that control the activity of other genes,” Dr. Ambros notes. “The challenge is to find out what are the in vivo targets for all of the microRNAs, and what is the importance of their various regulatory actions for important biological processes.”
In a Family of Small Non-Coding RNAs
A member of a family of non-coding RNAs that include such meddlesome relatives as small interfering RNAs (siRNA), and short hairpin RNAs (shRNA), miRNAs were salvaged from regions of the genome that at one time were referred to as junk DNA, because they consist of stretches of bases that do not encode proteins. However, despite being plentiful, Ambros notes that all non-coding RNAs represent only a small percentage of the long stretches of DNA bases that lie between protein coding genes,” notes Dr. Ambros.
“Much of the junk DNA is retrotransposons,” he says.
The entire family of non-coding RNAs began to be looked at closely after 1998, the same year that Craig Mello and Andy Fire published their landmark paper about the process of RNA interference (RNAi), in which double-stranded RNA silences homologous genes in an antisense fashion. A cousin of miRNAs, siRNAs are an intermediate in RNAi (2).
MicroRNAs differ from siRNAs and shRNAs in both the scope of target mRNAs they interact with and in the mechanism of how they interfere with the translation of target mRNAs into proteins. In fact, one reason that miRNAs intrigue researchers like Dr. Ambros is that, in animals, they often repress translation of target mRNA molecules rather than silence them through directed cleavage of the transcripts.
“There are a few hundred microRNAs in the human genome,” Dr. Ambros continues, “and there may be a few hundred other miscellaneous non-coding RNAs that have yet to be discovered.”
The characteristic hairpin structure of the ~70 nucleotide pre-cursor miRNAs has helped researchers to discover hundreds of different miRNAs throughout many different cell types. To find many of these miRNAs, researchers have used computational methods that rely on structure-based informatics searches of non-coding genomic sequences.
“We can easily recognize protein-coding sequence because we know the genetic code, but since we don’t have the salient features of all non-coding RNAs, it’s hard to find them. But, for microRNAs, we now know that they make a hairpin structure, and so it is becoming easier to find them with computational means,” says Dr. Ambros.
Defining Cell Fates
Some of the biological processes for which Dr. Ambros’s lab has already characterized microRNAs include developmental timing processes in C. elegans. Through the study of worm and fly mutants, Dr. Ambros’s lab hopes to learn how these miRNA genes regulate biological pathways involved in the development of these animals.
Like lin-4, the let-7 (lethal-7) gene also encodes a miRNA that represses translation of mRNAs that encode proteins of the heterochronic timing pathway of C. elegans. Worms that do not have the let-7 gene do not pass from the last larval stage into adulthood.
“In addition to lin-4 and let-7, three other let-7 related miRNA genes are functioning in this timing pathway,” says Dr. Ambros. “And, there’s also a second lin-4 related miRNA gene that’s also contributing to the timing pathway.”
Dr. Ambros’s lab discovered how some of these let-7-related miRNAs function in fly and worm development through a technique known as reverse genetics. In reverse genetics, a researcher identifies the function of a gene by removing or knocking out that gene, which creates a mutant organism that lacks the gene. The researcher can then learn about the function of that gene based on changes in behavior or appearance of the mutant missing the gene.
“Many mutant genes have been identified in the nematode timing pathways, so, by working on the genetic relationship amongst the genes, we can figure out which ones are regulating which other ones,” notes Dr. Ambros.
A detailed understanding of miRNA targets in worms and flies will also help other researchers determine if similar targets are conserved in the analogous pathways in humans and mice.
Dr. Ambros acknowledges that it’s difficult to predict the transfer of scientific knowledge between worms, flies and humans. He notes, however, that a complete understanding of how stage-specific fates in flies and worms are controlled at the molecular level may lead to analogous experiments that uncover timing and development pathways in human stem cells.
Refining Cell Types
Not only do miRNAs play a significant role in how cell types are defined in the development of flies and worms, but they have also been found to have a more subtle influence on cell development, the refining of cell types. For example, Dr. Ambros cites what he refers to as some fascinating research that has recently been published by Oliver Hobert’s lab at Columbia University.
In August 2004, Dr. Hobert published a paper in Nature describing the roles of microRNAs in establishing left-right asymmetry for a subclass of sensory neurons in C. elegans. In this finding, the neuronal cell type is established independently of the microRNAs, but, to make the left side of the cell different from the right side, two different microRNAs are expressed sequentially that interfere with mRNA transcripts for transcription factors that regulate expression of genes specific for left and right chemoreceptors. As a result, the left and right cells express different chemoreceptor genes (3).
“In this example, microRNAs act to refine cell type specification in the nervous system,” says Dr. Ambros.
Dr. Ambros adds that miRNAs may participate in this same kind of refining of cell types in the vertebrate nervous system.
Another example of how miRNAs refine cell types occurs in the muscle cells of the fruit fly Drosophila. In unpublished work, Dr Ambros says his lab has found at least one miRNA involved in the proper development of muscle in the fly. The lab has found that, in mutant flies that are missing the miRNA, the muscles are formed, but their morphogenesis is drastically abnormal, and the fly larvae die.
“This [finding] suggests that this microRNA regulates some sort of pathway or pathways in muscle that are vital for the morphogenesis of the tissue,” he says.
Real-Time PCR Assays Detect and Quantify MicroRNAs
To strive for a more comprehensive understanding of how microRNAs contribute to important biological processes, Dr. Ambros’s lab has begun to detect and quantify miRNAs using a sensitive and specific assay from Applied Biosystems based on real-time PCR technology. He hopes to use these soon-to-be released real-time TaqMan PCR assays from Applied Biosystems to reveal some of the various roles that miRNAs play in regulating gene expression and determining the fate of different cell types in a variety of organisms.
The TaqMan-based real-time PCR assays use stem-looped primers that enable a two-step quantification of miRNAs present in a sample. In the first step, stem-looped primers anneal to target miRNAs and extend the length of the molecule by reverse transcription PCR (RT-PCR). In the second step, a real-time PCR reaction that involves a reverse primer and a probe enables researchers to quantify the number of mature miRNA molecules present in a sample based on fluorescent emission of a reporter dye.
One of the hallmarks of the TaqMan-based real-time PCR assays is their ability to distinguish between the hairpin structure of pre-cursor miRNA and the short mature miRNA molecules. The stem-loop structure, which is specific to the 3’ end of the mature miRNA, presumably creates steric hindrance to prevent priming of the pre-cursor miRNA. As a result, the assays detect and quantify only mature miRNA molecules, the form capable of interacting with target mRNA molecules.
“The kinds of assays that we perform are trying to measure RNA levels, both of the target mRNAs and of the microRNAs themselves under different developmental conditions or genetic backgrounds,” explains Dr. Ambros.
Until participating in a recent collaboration with Applied Biosystems to investigate the use of real-time PCR assays for detection and quantitation of microRNAs, Dr. Ambros had been primarily using Northern blotting to measure RNA levels of both mRNAs and microRNAs.
In Northern blotting, target RNAs are detected by complementary DNA or RNA probes that hybridize to them.
Northern blotting, while useful for detecting the presence of RNA in samples, is slower, requires more initial sample, and does not generate quantitative data such as those resulting from real-time PCR assays that use TaqMan probes and primers.
“The TaqMan data are much better than the Northern data in accuracy, sensitivity, and reproducibility. The sensitivity, in particular, is vastly superior to Northern blots. The size of sample that one needs is several orders of magnitude smaller in quantity, and with that small sample, one can still profile all of the known microRNAs. This is a major jump in technology,” says Dr. Ambros.
The ability of the TaqMan-based real-time PCR assays to accurately quantify miRNA levels will provide data that more accurately describe the role of miRNAs in determining the fates of cells.
Quantification of miRNA levels will enable researchers to compare wild-type and mutant nematodes and see more clearly how expression of specific miRNAs regulate the expression or function of other genes.
“What’s important about the accuracy and the sensitivity of the TaqMan assay is that very often we find that two-fold or three-fold changes in the level of a regulatory molecule can actually have very significant effects. This realization has meant that we are often frustrated with methods such as Northern blots, where we can’t really be sure if the level of the RNA has changed significantly,” says Dr. Ambros.
Finding MicroRNA Targets
Another major advance in developmental biology research that Dr. Ambros anticipates will be made possible by real-time PCR technology will be the lab’s ability to work with small samples.
He believes that the extreme sensitivity of the TaqMan real-time PCR assays will make it possible to manually collect very small samples of staged embryos of the fly or the worm and then assay levels of specific miRNAs in those samples.
“Right now, we need many micrograms of RNA for Northern blots, and so, therefore, we have to grow many large cultures, even then it’s difficult to achieve accurate staging. It’s cumbersome, and slow to generate these accurately staged samples for developing animals. Now, however, we can do that,” he says.
“We also need to know, in worms, when do microRNAs first appear in certain cell lineages. We can assay when their promoters are active, but we don’t know when the mature microRNAs first appear, so, for this, the TaqMan [technology] is now the most sensitive assay that we have available,” notes Dr. Ambros.
The ability to assay small samples for miRNAs, will also benefit the Ambros lab’s studies of the development of muscles in the fly.
There is still much to be learned about how miRNAs interact with target mRNAs before it will be possible to understand the exact nature of the regulatory roles that miRNAs play in animals.
Loose Fit to Targets Represses Expression
Often, researchers use computers to predict potential miRNA targets, based on sequence matches. They then perform experiments to test whether the predicted targets exist in-vivo. However, there are limitations to using computer programs to find miRNA targets.
“MicroRNAs are so loosely matched to their targets that statistically the computer can’t be very confident about the target predictions. The field is not really settled on a reliable consensus for how one finds targets computationally,”says Dr. Ambros.
According to Dr. Ambros, in animals, miRNAs are thought to bind to target mRNAs primarily in the 3’ untranslated region (UTR). However, in almost all interactions with a target mRNA, the miRNA does not bind in a tight base-to-base pairing, such as occurs between the two complementary strands of DNA in a double-helix.
“The fact that the base pairing is incomplete is one of the hallmarks of essentially all animal microRNA targeted interactions. There is mismatch base-pairing, and apparently it occurs predominately in the 3’ UTR.”
This loose-fitting binding means that the target mRNA molecule is not destroyed, but that the process of converting the mRNA message into protein sequence is instead repressed.
According to Dr. Ambros, differences in how plant and animal miRNAs interact with their targets implies different mechanisms for how miRNAs interfere with translation in the plant and animal worlds.
“In plants, most often microRNAs do interact with the coding region of target mRNAs, and, moreover, they match their targets precisely. This results in target destruction, instead of translational repression. There’s something about translational repression, which is a common mechanism employed in animals,” explains Dr. Ambros.
Dr. Ambros envisions future experiments that involve quantifying miRNA levels that may help explain the significance of miRNA repressing translation of mRNA transcripts instead of cleaving and destroying them.
“A hypothesis that I’m really keen on testing is the idea that the microRNAs provide a rapid response that could be modulated by signals, which might be affecting the abundance of the microRNA or its efficacy. If you had a reversible translational repression then it may be more responsive then other modes of regulation,” he says.
What has been shown is that miRNAs can interact with target mRNAs to reduce rather than turn off expression of particular mRNA transcripts into related proteins.
Although it has yet to be shown experimentally, Dr. Ambros believes that there may be a correlation between the number of miRNAs and the number of target mRNA sites, where an interaction would repress translation. “If you have fewer sites, and less of the microRNA, it would produce an intermediate level of repression,” he says.
Many questions still remain about how miRNAs interact with target mRNAs, or even other potential targets. However, the ability to accurately quantify both mRNA and miRNA molecules should help to answer some of these questions. For example, although currently there is no experimental evidence of a correlation existing between the degree of target mRNA translational inhibition and the number of miRNA molecules bound to a target, Dr. Ambros notes that this is an operating hypothesis of the field and “There’s experimental evidence that the number of sites in a UTR can effect the potency of repression.”
Moreover, Dr. Ambros asserts that assays that enable accurate quantitation and sensitivity will provide researchers with new insights about functional cooperation between miRNAs and their targets.
“For any question of function,” he continues, “where it becomes important to know whether or not [a particular] miRNA is present, or whether or not its level changes based on different conditions, those would be the questions for which this TaqMan assay would be very useful.”
MicroRNAs and Brain Cancer Research
While miRNAs are expressed in many different human cell types, according to Dr. Ambros, the brain cells express the greatest variety of microRNAs.
In addition to their studies on developmental pathways in worms and flies, Dr. Ambros’s lab is in a collaboration with Dr. Mark Israel, an oncologist, who heads the Norris Cotton Cancer Center at Dartmouth. Together, the two scientists have been investigating ways to use data about expression of miRNA genes as a means for distinguishing between two different kinds of brain tumors, both are astrocytomas found in one type of glial cell.
“We are profiling microRNAs in brain tumors, and in cell lines derived from human brain tumors because certain classes of these tumors are not as responsive as other classes are to treatment,” notes Dr. Ambros.
Because the prognosis for recovery in the class of tumors responsive to treatment is much better than it is for the nonresponsive class of tumors, Dr. Israel and Dr. Ambros are searching for a method or technique that can reliably distinguish between these two types of tumors. Traditional pathological assays and histological approaches have not been useful for distinguishing between these two types of tumor cells.
Dr. Israel, however, has had some success distinguishing among the different tumor types by using microarrays to profile these brain tumors. However, microarrays cannot provide the same kind of quantitative data that real-time PCR assays generate.
Because miRNAs are particularly prevalent in brain cells, the two researchers have been investigating the possibility that miRNAs expressed in these tumor cells may serve as biomarkers that may indicate whether a particular tumor cell is the type that is more responsive to treatment.
In previous work, Dr. Ambros’s lab profiled miRNA levels in mouse and human cell lines that were neuronal carcinoma cells and cell lines capable of being induced to differentiate. They wanted to see if there was some correlation between neuronal differentiation and miRNA expression. They found that miRNAs were brain specific across mouse and human (5).
“But the method that we used for that study was Northern blots, and it’s very low throughput, labor-intensive, and expensive. When Dr. Caifu Chen, [Senior Staff Scientist, Applied Biosystems] contacted us about using the TaqMan assays we were very interested.”
Results from the TaqMan real-time PCR assays of different brain tumor types have shown, in some cases, a consistent pattern of miRNA expression across tumors and cell types. According to Dr. Ambros, other miRNAs behave quite differently in different tumors
“This has been a terrific collaboration between Applied Biosystems, and us,” Dr Ambros continues, “because Mark has provided the samples and Caifu has performed the profiling using the high-throughput Applied Biosystems real-time PCR machines. He then sends us the data. What we can see already is that some of the microRNAs are dramatically up-regulated or down-regulated in the astrocytomas.”
“We see signatures that may turn out to reflect important biological properties of these tumors. So, we’re excited about profiling more tumors,” notes Dr. Ambros.
Next, researchers in Dr. Ambros’s lab plan to use bioinformatics tools to relate the pattern of changes in miRNA to those sets of predicted targets that are either present or absent in profiled tumors.
Connecting MicroRNA Levels with Cell Fate
For Dr. Ambros, the next stage of miRNA profiling that his lab plans to pursue involves correlating expression profiles of miRNAs with those of mRNAs of potential target genes in the same cell.
TaqMan real-time PCR assays that quantify miRNAs give researchers like Dr. Victor Ambros a powerful tool for detecting and quantifying levels of miRNAs in different cell types. Use of these assays will enable researchers to connect expression of miRNAs with regulatory processes that ultimately determine the fates of many different cell types. For example, in the case of heterochronic pathways, and left-right asymmetry in the nervous system in C. elegans, muscle development in Drosophila, or even tumor formation in human brain cells, whether or not specific microRNAs are expressed in a cell often influences key cellular characteristics and functions carried out by that cell.
References
- Lee, R.C., Feinbaum R.L., and Ambros, V., “The C. elegans Heterochronic Gene
lin-4 Encodes Small RNAs with Antisense Complementarity to lin-14,” Cell Vol 75: 843-854, (December 3, 1993). [Medline abstract]
- Fire, A., et al. “Potent and Specific genetic interference by double-stranded RNA in Caenorhabditis elegans.” Nature 391, 806-811 (1998). [Medline abstract]
- Chang S, Johnston RJ Jr, Frokjaer-Jensen C, Lockery S, Hobert O., “MicroRNAs act sequentially and asymmetrically to control chemosensory laterality in the nematode,” Nature 430 (7001):785-789, (Aug 12, 2004). [Medline abstract]
- Livak et al. “Oligonucleotides with Fluorescent Dyes at Opposite Ends Provide a Quenched Probe System Useful for Detecting PCR Product and Nucleic Acid Hybridization,” Genome Research 4: 357-362 (June, 1995).
- Sempre, L.F., Freemantle, S., Pitha-Rowe, I., “Expression Profiling of Mammalian MicroRNAs Uncovers a Subset of Brain-expressed MicroRNAs with Possible Roles in Murine and Human Neuronal Differentiation,” Genome Biology Vol 5, Issue 3: R13, (February 16, 2004). [Medline abstract]
Trademarks
For Research Use Only. Not for use in diagnostic procedures
The PCR Process and 5’ nuclease process are covered by patents owned by Roche Molecular Systems, Inc. and F. Hoffmann-La Roche Ltd.
Applied Biosystems is a registered trademark of Applera Corporation or its subsidiaries in the US and/or certain other countries.
TaqMan is a registered trademark of Roche Molecular Systems, Inc.
|
