RNA, microRNAs and human disease

In the 1980s, molecular geneticists began trawling the human genome for mutant genes involved in inherited disorders. The approach proved fruitful, but sometimes, highly prospective logarithm of the odds (LOD) scores led nowhere - at least, not to mutant alleles of suspect genes.

Less than a decade ago, biology's central dogma held that genes encoded in DNA are transcribed into messenger RNAs, which are then translated into protein. "So much for the simplified view," Professor Scott Hammond told the 2007 Lorne Cancer Conference.

"It's now apparent that a large fraction of the genome doesn't code for known genes. Recent data suggests at least 50 per cent of the genome is transcribed into RNA."

The University of North Carolina molecular geneticist was making his second appearance at Lorne in 12 months, to update his 2006 report on his research team's discovery of a cancer-causing microRNA - an oncomiR -called chr13orf 25 (chromosome 13 open-reading frame 25).

If more than 50 per cent of the genome codes for RNA molecules that collectively organise and regulate the complex activity of the genome, the implication is that many hereditary disorders may ultimately arise in the 'R-nome', and involve dysregulation of the activity of perfectly normal genes - or entire gene networks.

microRNAs are tiny molecules, of 23 nucleotides or fewer, cleaved from larger precursor RNA transcripts encoded within introns, or the vast tracts of DNA between genes. The function of most microRNAs is unknown.

Why did nobody detect them before? Because when researchers ran cellular extracts of messenger RNAs on electrophoresis gels, the tiny microRNAs migrated rapidly to the ends of the gels, and fell off the edge of the known RNA world.

The single-stranded precursor RNAs, typically hundreds of bases long, fold back on themselves and undergo complementary base-pairing to form double-stranded 'hairpin' molecules that are cleaved into multiple, functional micro-RNAs.

"Micro-RNAs are a distinct class of non-coding RNAs that work by blocking gene expression," Hammond says. "They have been discovered in many different organisms - more than 400 have been found in the human genome.

The data suggest that microRNAs regulate a large fraction of the genes in the human genome."

MicroRNA expression

MicroRNAs appear to play key roles in coordinating embryonic development in organisms as disparate as nematode worms, fruit flies, plants, zebrafish and humans.

Among other things, they regulate the timing of the cascade of developmental events after conception. In Drosophila, microRNAs regulate the cell cycle, cell growth and differentiation and apoptosis.

Their role in transforming stem cells into terminally differentiated cells led Hammond and his colleagues to wonder if mutation-induced microRNA regulatory errors might have a causal role in cancer, which arises from disruption of the normal processes of cellular differentiation and apoptosis.

Do microRNAs promote or suppress tumorigenesis? Are they involved in regulating cell proliferation? "Imagine a microRNA that regulates a tumour-suppressor gene like P27, which stops cell proliferation - if it is mutated, it might then promote cell proliferation," Hammond says. "Or perhaps microRNAs regulate apoptosis, by acting on pro-apoptotic genes."

Hammond's laboratory developed its own RNA microarray platform to compare microRNA expression patterns in cancerous and normal cells. "We can now [quantify] microRNA expression in individual cancer tissue samples," he says.

"We've also looked at normal microRNA expression patterns from early to late embryonic development in the mouse, and in the adult mouse. We have identified a large group of microRNAs that are highly expressed in adult tissues, and others that are expressed at high levels early in development, then decline.

"MicroRNAs not expressed early are elevated in mid to late development, then expressed at very high levels in the adult mouse. Comparing microRNA expression in tumour cell lines with expression in normal adult tissues, we found that the patterns are reversed: microRNAs expressed at high levels in differentiated tissues are shut off in cancerous tissues. So cancer microRNA expression patterns resemble expression patterns in early embryonic development."

Cause and effect

But Hammond says the microRNAs may not actually cause the cancer - their expression is altered in cancerous tissues. Hammond's team searched for a cause-and-effect relationship using their 13-ORF24 oncomiR as a model.

Endonuclease cleavage of the microRNA cluster yields seven microRNAs, which Hamond's team found were overexpressed in a mouse model of lymphoma. Moreover, the over-expressed microRNAs dramatically accelerated the cancer's development, suggesting that re-expression of 'early-on' micro-RNAs promotes tumorigenesis.

To determine what causes over-expression of the chromosome 13 microRNA cluster in cancer, Hammond and his colleagues investigated how its transcriptional is regulated. They identified the core promoter region and showed that it is regulated by E2F transcription factors. This, in cooperation with the c-Myc oncogene, which drives cell proliferation, would cause high expression in tumour cells.

Hammond's team has also investigated how cancerous cells inactivate at least 100 microRNAs normally found in differentiated cells, in a seemingly coordinated manner, by monitoring one microRNA, Let-7g, during normal embryonic development.

Let-7g is normally induced midway through embryogenesis, as part of a long primary transcript. The endonuclease Drosha, part of the cell's RNAi machinery, cleaves it to form a 79-nucleotide hairpin RNA that is exported to the cytoplasm.

The endonuclease Dicer then excises the loop of the hairpin, creating the active, 21-nucleotide microRNA.

The Let-7g microRNA is expressed at a very low level in early embryogenesis, so Hammond's team was surprised to discover that the long, primary transcript is expressed uniformly throughout embryonic development, while the embedded Let-7g microRNA does not become abundant until mid-development.

"Clearly, something blocks processing at the Drosha cleavage stage," he said. Other mid- to late-on microRNAs follow a similar pattern of expression. The question is whether they are regulated by the same mechanism.

"As the cells begin to differentiate, we see a release of Drosha, and large amounts of processing to release mature microRNAs. So something must be blocking Drosha in early embryogenesis. If you take the primary transcript and put it into a human nuclear extract [from differentiated cells] you get processing and mature transcripts."

MicroRNA regulation

Large data set are already available on microRNA expression patterns in tumour cells, showing that around 25 per cent of all microRNAs are regulated in the same way.

"Tumour cells have a global reduction in microRNA expression, relative to differentiated cells. So microRNAs are not being regulated at the transcriptional level, but during processing - the cell reduces expression of mature RNAs without regulating expression of the primary transcript."

Reducing Drosha causes a reduction in mature micro RNA expression. So what regulates Drosha? And following from that question: could a targeted therapy to activate Drosha in tumour cells force them to differentiate and die?

Watch this space. Lorne Cancer Conference organisers probably already have Hammond high on their list of must-have speakers for 2008.