Op-shopping the genome yields RNA gold

Shakespeare’s Hamlet was correct - “what a piece of work is a [hu]man! ... how infinite in faculty” - and now it seems, according to a team of researchers in Sydney, in gene expression regulation.

Professor John Rasko established the Gene and Stem Cell Therapy Program in 1999 at the Centenary Institute in Sydney. These days the program comprises around 20 researchers whose work spans genetics, stem cells, gene transfer technologies, as well as RNA biology and cancer.

The work that Rasko will discuss at the Lorne Cancer (and Genome) meetings (and published recently in Cell) started about 5 years ago and is based on over a decade of research aimed at better understanding small non-coding (snc) RNA molecules and their potential targets in white blood cells (granulocytes).

A puzzle to be solved

Until relatively recently, non-coding nucleic acid sequences all fell into the ‘junk’ category of genetic information, despite their abundance in genomes and conservation across species. But in light of the many studies revealing this assumption to be false, not least of which the microRNA story and Rasko’s own work, his team set out with a firm hypothesis about the role of non-coding RNA molecules and gene splicing mechanisms in white blood cell development (granulopoiesis). They then proceeded to establish all of the pieces needed to solve their particular puzzle.

“We amassed a vast amount of data - from RNA sequencing to the whole suite of ‘omics’ - plus a full bioinformatics toolkit,” recalled Rasko.

However, as so often happens in science, their initial hypothesis turned out to be completely wrong. Having invested significant time and money, they turned to a back-up plan. The result was a nifty new computer algorithm that allowed them to totally reanalyse their data and explore some fairly intractable questions about the role of introns in granulocyte differentiation. And basically, they hit gold by uncovering a completely unimagined mechanism of gene expression regulation.

According to Rasko, it really was an ‘OMG!’ moment. Dr Justin Wong with his background in epigenetics and molecular biology then set about devoting the next two years to prove the idea with other members of the group.

More than one way to splice a gene

So why look at introns? According to Rasko, mystery has always surrounded these parts of the genome.

Introns are highly conserved, non-coding regions that sit among the gene exons, which are those bits of the genome that are eventually translated into proteins and, thus, long regarded the most important genetic components of cells. By looking at the non-coding regions of genomes, Rasko and his team have found that introns can and do control the expression levels of perhaps hundreds of genes that are differentially expressed during the development of white blood cells of the innate immune system.

During gene transcription, the introns in most eukaryotic genes are removed from pre-messenger RNAs by the cell’s splicing machinery to produce protein-translatable mature mRNAs. However, the introns are sometimes retained as part of the mature mRNA sequence by an alternative splicing mechanism called intron retention (IR).

Most IR is thought to reflect cellular ‘mistakes’, often due to splicing mutations, that are dealt with by a cell’s garbage collection machinery. However, RNAs transcribed from intronic regions have been implicated in a number of processes related to the post-transcriptional control of gene expression and are tipped to regulate gene expression in viruses and some plants.

How IR plays a role in normal gene expression, which tissues are affected and why it happens remains largely unknown.

The white blood cell model

The final phases of granulocyte maturation in the bone marrow - from promyelocyte to myelocyte to mature granulocyte - are marked by distinct transcriptional and translational changes, accompanied by little-understood changes in the shape and size of the cell, especially the nucleus. These unique cellular stages can be easily isolated based on specific cell-surface markers and thus provide a valuable and well-studied model for cellular differentiation.

Switching questions was therefore an easy decision for Rasko because it turns out that granulocytes also have quite a lot of IR going on. In fact, removal of transcribed intronic material via the cell’s nonsense-mediated decay (NMD) pathway is somehow essential for the normal development of haematopoietic stem and progenitor cells.

Rasko’s group was therefore keen to examine why these cells retained so many introns and whether this phenomenon was functionally relevant to the specific and complex steps of granulocyte maturation.

Finding the baby in the bath water

To approach this slightly different focus, the researchers realised that by the very nature of RNA sequencing technology and software, much of the information they might need about introns is actually thrown out. And, according to Rasko, their eventual ‘ah-ha’ moment was a direct consequence of this realisation.

“When we do deep RNA sequencing, we basically ask the machine to analyse an immense catalogue of relatively short bits of sequence and match them back to unique locations on a reference genome to create the sequence map,” said Rasko.

However, introns often contain degenerate or low-complexity stretches that are, statistically speaking, harder to match to a unique site in the genome. To deal with the zillion bits of data involved in sequencing a whole genome, the machine therefore has to preferentially ditch most of the sequencing data that is specific for introns.

“To get around this, Dr William Ritchie developed and applied a novel and very rigorous computer algorithm, called IRFinder, to extract information solely about introns from all the data we had already collected for our different granulocyte cell populations; hopefully to pinpoint where the introns are being retained,” explained Rasko.

“And that is the moment when we knew we had something big, because there were all these examples, almost 100 genes even conservatively, that were retaining introns and being affected functionally and differentially by IR during normal granulopoiesis in terms of reduced protein expression. We knew we had uncovered a major mechanism of gene control that was previously ignored because people basically could not see the data for this low-complexity stuff.

“Finally, Drs Jeff Holst and Chuck Bailey in the lab showed that by perturbing IR specifically in a gene called Lmnb1, which shows a very high level of intron retention, we saw a dramatic alteration in nuclear morphology and cell numbers,” Rasko continued.

“The result was unexpected and very exciting because it suggested to us that, in contrast to the prevalent view that such mechanisms are limited to mRNAs encoding aberrant proteins, IR is a critical regulatory pathway programmed into the cell to operate during normal granulocyte development. This was not some accident or some failed intron excision … these are highly specific introns in particular genes. And it is occurring when there is no disease state or mutation.”

Adding weight to this importance was the team’s subsequent finding that the genes affected by IR in granulopoiesis are conserved between mouse and human.

Lots more to learn

“We are obviously very excited about this work and certainly pursuing IR in this context to explore how broadly conserved it is. Just when did nature invent this mechanism of gene expression control? And what other cell and tissue types are affected by IR as a normal part of gene expression, including how early in differentiation does it appear … stem cells for instance?

“Also, Why some introns or genes and not others? What molecular markers are present in those genes that cause the splicing machinery to ignore them in an apparently normal physiological process? What sets it off in the first place remains an interesting and open question.”

Rasko already knows from their work and that of collaborators that IR also affects lymphoid cells, and probably neuronal cell production. Moreover, it is very likely to be aberrant in disease states such as leukaemia and thus may offer new therapeutic targets - indeed, this is the subject of a new NHMRC grant for Rasko’s group.

“If you had told me five years ago that we were going to discover a completely new mechanism of gene expression regulation in mammalian cells I would have laughed,” said Rasko, about the possibility of yet more to discover.

“A fantastically dedicated local team along with our collaborators and funding from diverse sources have revealed that previously unimagined mechanisms are ripe for discovery … if only we keep an open mind! And we should keep looking for even more!”

**********************************************************

Lorne conference line-up

Here’s the line-up for the Lorne conferences for 2014, to be held at Mantra Lorne on the Victorian south coast.
19th Lorne Proteomics Symposium
February 6-9
http://www.australasianproteomics.org/lorne-proteomics-symposium-2014/
39th Lorne Conference on Protein Structure and Function
February 9-13
http://www.lorneproteins.org/
26th Lorne Cancer Conference
February 13-15
http://www.lornecancer.org/
35th Lorne Genome Conference
February 16-19
http://www.lornegenome.org/
Lorne Infection and Immunity
February19-21
www.lorneinfectionimmunity.org

**********************************************************

Professor John Rasko AO is a clinical haematologist and director of Cell and Molecular Therapies at the Royal Prince Alfred Hospital in Sydney and heads the Gene and Stem Cell Therapy Program at the University of Sydney. He studied medicine at the University of Sydney and completed a PhD at the WEHI under Professor Don Metcalf and Dr Glenn Begley in molecular haematology. He then pursued postdoctoral research in gene therapy at the birthplace of bone marrow transplantation: the ‘Hutch’ in Seattle. His work in gene therapy, experimental haematology and cell biology has led to discoveries involving new mechanisms of leukaemia, blood hormones, stem cells RNA biology, amino acid transport and clinical trials of new therapies for cancer and bleeding disorders.

Image credit: ©iStockphoto.com/Eraxion