RNAi: A Taste of Cellular Asymmetry
The regulatory machinery governing gene expression includes proteins called transcription factors, which interact directly or indirectly with genomic DNA.
Published June 1, 2005
By Beth Schachter, PhD
Academy Contributor
In complex organisms, what distinguishes one cell type from another? For example, in mammals, what sets apart liver cells from pancreas cells? Most prominently, liver cells churn out proteins such as albumin. Pancreas cells make and secrete a different set of proteins, including insulin.
Such organ-specific protein production comes about during early development of the organism. This differentiation process involves both activating and inactivating groups of genes in cell-type specific patterns. The regulatory machinery governing gene expression includes proteins called transcription factors, which interact directly or indirectly with genomic DNA. This interaction between transcription factors and specific genes in the DNA leads to a change in the rate of RNA transcription from those genes.
RNA Interference (RNAi)
Transcription factors are not the only macromolecules that regulate gene expression, i.e. that govern transcription of DNA into messenger RNA and subsequent translation of messenger RNA into protein. Recently, scientists were surprised to find that certain classes of RNA molecules have regulatory roles as well. Unlike transcription factors, which elicit both stimulatory and inhibitory effects, regulatory RNAs seem to be just inhibitory. Consequently, this process is called RNA interference (RNAi). The discovery of RNAi created such a stir that, in 2002, Science Magazine dubbed it the breakthrough of the year.
One class of RNAi molecules is that of the microRNAs (miRNAs), so-called because of their small size. miRNA derivatives team up with specific proteins to form RNAi complexes that inhibit messenger RNA translation into proteins. This translation inhibition is sequence-specific; the miRNA base pairs with its complementary sequence in messenger RNA (mRNA) molecules.
Many of the RNAi pioneers work in or near New York City. Therefore, when The New York Academy of Sciences (the Academy) initiated its Frontiers of Science Discussion Groups, the RNAi Group was among the first and has proven to be consistently vibrant. Highlights from a talk at the October 2004 meeting, Frontiers of RNA Silencing Mechanisms, offered a window into this newly discovered biological regulatory mechanism used by animals and plants alike.
Control of Neuronal Development
Columbia University neuroscientist Oliver Hobert set out to study the origin of brain asymmetry. As Hobert explained, the brain – whether in a human or a worm – is bilaterally symmetric. But, within this apparent anatomical symmetry are distinct bits of functional asymmetry.
Take neural control of language in humans. It is highly asymmetric, and the functional asymmetry comes about during development. An embryonic brain seems to have complete mirror-image symmetry, and yet it evolves into a structure in which certain functions are lateralized.
Shining a Light
What molecular mechanisms enable asymmetry to evolve from mirror-image symmetry? Hobert looked for a simple system in which to study this question. He chose the brain of the nematode Caenorhabditis elegans, focusing on a pair of neurons, dubbed ASE right (ASER) and ASE left (ASEL), involved in taste.
Inspection of these two neurons in adult brains showed identical input and output connections with other cells. ASEL senses sodium, however, while ASER senses chloride.
The worm’s lateralized sodium/chloride discrimination is tightly linked to the presence of one or another membrane protein. In adult worms, the ASER makes the protein gcy-5; ASEL makes the protein gcy-7. ASER does not make gcy-7, and ASEL does not make gcy-5, implying that this asymmetry involves both on and off switches.
To study regulation of gene expression within individual neurons, researchers often use transgenic animals – engineered so that the promoter (the cell-specific control region) of the gene of interest drives expression of a marker protein, such as green fluorescent protein (GFP). Hobert and his group used just such hybrid constructs. They linked the gcy-5 or gcy-7 promoter to GFP, for example, to make transgenic worm lines of each sort.
When the researchers inspected the wild-type transgenic worms, looking for green fluorescent cells, they learned that, in the embryos, both ASEL and ASER express gcy-7 but not gcy-5. This implies that, later in development, switches in ASER turn gcy-7 off and gcy-5 on.
Scoring the Mutants
The researchers then randomly induced mutations to create transgenic stocks. Included among the mutant worms were some that inappropriately expressed either the left- or right-cell fate marker in both ASER and ASEL. Still other mutants expressed neither fate marker. By determining the nature of the mutation in each of the mutant worms, Hobert and his group started piecing together the puzzle of how the ASEL/ASER asymmetry arises.
These studies showed that proper differentiation of both ASE cells – the ASE “ground state” – requires expression of the transcription factor che-1 early in development. che-1, in turn, activates several additional genes in the ASE cells. The hunt was now on for factors that act downstream of che-1 to switch gcy-5 on and gcy-7 off in ASER but not in ASEL.
Hobert’s group turned up several additional players that make up a feedback regulatory cascade. In the adult brains, some components are expressed bilaterally in the ASE neurons and some are expressed asymmetrically.
Another Transcription Factor
Most notably, along with inducing several transcription factors, che-1 indirectly induces lsy-6, (pronounced lously 6). Surprisingly, the product of the lsy-6 gene is a miRNA, and not, as expected, another transcription factor.
lsy-6 is the first miRNA shown to play a role in nervous system development. The Hobert group wasn’t looking for miRNAs in their hunt for determinants of the ASEL/ASER phenotype. In fact, part of the power of their functional screening strategy is its absence of bias toward identifying just protein-coding genes. Instead, it can identify different types of regulatory factors, including genes for non-protein-coding regulatory RNAs.
Having discovered the lsy-6 miRNA, the Hobert group looked for its target, i.e. a messenger RNA that interacts with lsy-6 and, therefore, is not translated into protein. That target turned out to be the messenger RNA for cog-1, another transcription factor in the cascade; one needed for the ASER phenotype.
Further work showed that the regulatory cascade contains at least one other miRNA. Thus, the newly identified miRNAs are part of a regulatory loop including both transcription factors and miRNAs as regulators of cell fate.
Functionally Equivalent Mechanisms
These results led Hobert to propose that, as developmental regulators of cell fate, miRNAs and transcription factors are functionally equivalent switches of gene expression.
As he explained, transcription factors interact with specific sequences in and around genes, called the cis-acting regulatory sequences. Typical genes have multiple cis-acting regulatory sequences. The determination of when, where, and how much the gene will be transcribed in any given cell depends on the combination of transcription factors active in the cell.
Evidence from Hobert’s lab and elsewhere suggests that translational regulation by miRNAs also involves multiple miRNAs acting on individual mRNAs, perhaps in a cooperative manner. Therefore, whether a given mRNA is or is not translated may depend on the repertoire of miRNAs that a particular cell type expresses.
While transcription factors regulate transcription and miRNAs regulate translation, Hobert concludes, both influence which proteins will be made in any cell at a given time. They thus use functionally equivalent mechanisms, Hobert argues.
The story is still far from final. Hobert’s findings identify a regulatory loop that governs the ASEL/ASER asymmetry, but they don’t yet show what the initial input to and output from the loop are.
Hobert already sees clues, however, to the input – the primary event that establishes the laterality of ASEL and ASER function is the input. He notes that the critical event may happen in early embryogenesis, but not be revealed until much later in development. He suggests, for example, some sort of epigenetic modification of chromatin, which would be functionally dormant until much later. Studies in the Hobert lab are starting to address this key question.
Charting the RNAi Frontiers
Hobert’s chronicle of a regulatory role for miRNAs in neural development typifies the talks at the RNAi Discussion Group events in being at the frontier of this new research field. Also that evening, Markus Stoffel (Rockefeller University) talked about a newly identified miRNA that regulates insulin secretion from the pancreas. As Stoffel explained, this finding may help identify new therapeutic targets for treating diabetes.
Finally that evening, Tariq Rana (University of Massachusetts) discussed his work on structural aspects of the RNAi machinery. He described his studies that determine which aspects of the miRNA structure participate in translation inhibition and which elements are dispensable. As he noted, that sort of information is key to developing useful new therapeutics.
Also read: The Research Behind Neurons and Cell Behavior
About the Author
Beth Schachter, Ph.D., is a science writer and editor in New York.
