Blanchette Lab

A molecular biologist “of a certain age” might misinterpret what Marco Blanchette, Ph.D., works on. He describes his interests as “how families of factors interact with their targets and co-regulate the same sets of genes. The source of this regulation is likely combinatorial, and we are teasing apart those combinatorial rules.”

Evaluating the global response and the impact of different stresses on processing of the expressed RNA

Image: Courtesy of Dr. Marco Blanchette

If it sounds like he works on transcription—the process that copies DNA into RNA—that’s a good guess, but wrong. Blanchette, a Stowers assistant investigator since 2006, studies gene expression from the perspective of RNA splicing, the process by which long stretches from a transcribed messenger RNA (mRNA) are snipped out to create diverse proteins from one gene.

Biologists have known for decades that RNA “filler” called introns are deleted from an mRNA transcript as it exits the nucleus, leaving behind only the protein-coding pieces, referred to as exons, to be decoded into protein. Early on, however, it became evident that RNAs originating from the same gene were not cut and pasted identically, as exons were often deleted or reshuffled in certain cellular contexts.

But it wasn’t until the human genome project revealed the deflating news that human DNA harbors only about 25,000 genes that the implications of alternative RNA splicing became clear—and face-saving—to Homo sapiens.

“People now know that there is little difference in the number of genes between humans and fruitflies, but we humans like to think that we are a little more complex than that,” says Blanchette. “That is the very importance of ‘alternative splicing’ of a particular pre-RNA—it enables you to generate many more proteins than the number of genes.”

Blanchette became interested in RNA processing as an undergraduate mentored by splicing expert Benoit Chabot at the Université de Sherbrooke in Québec. Confessing that he was initially “extremely bad at the bench,” Blanchette remained fascinated with how protein complexes recognize and know how to dice up an RNA strand, just because the process was so complex.

Receiving his B.Sc. in biology at Sherbrooke in 1994, Blanchette remained in the Chabot lab, where his project was to define how splicing was regulated in a single gene—the hnRNP A1 gene, which in itself encoded a splicing factor.

After “bashing the A1 pre-mRNA for sequences and factors that bind to those elements,” he published a study showing how a specific coding sequence, or exon, which is retained in some mRNAs, is skipped over and omitted when identical RNA-binding proteins bind to regions flanking it. That highly cited paper published in EMBO Journal in 1999 was the first to show how a specific protein directs alternative splicing mechanistically.

But Blanchette had spent six years understanding just one gene. By the time he earned his Ph.D. in 2000, he was ready to consider a more global approach to deciphering splicing rules. “By then microarray techniques had been developed to monitor overall changes in transcription but few people thought you could use those techniques to study alternative splicing,” he says.

However, Drosophila geneticist Don Rio at UC Berkeley was poised to take that plunge.  “The fly genome had just been sequenced. It was smaller than the human genome but its splicing was complex enough to be applicable to humans,” says Blanchette, “Berkeley was the one place all these ideas—genetics and genomics—were coming together.” He started post-doc’ing in the Rio lab in 2000.

Heat maps identify correlations among splicing regulators.

Image: Courtesy of Dr. Marco Blanchette

By 2005 Blanchette had developed a microarray platform to determine how populations of Drosophila pre-RNAs are processed. Pairing that technology with RNAi-based knockdown of four different splicing regulators he mapped the splice sites targeted by specific regulators. Overall he found that distinct classes of regulators recognized non-overlapping splice junctions—in other words, that global rules governing alternative splicing could be discerned.

In another analysis published with the Rio lab in a 2009 Molecular Cell paper, Blanchette examined interactions among hnRNP family proteins he had studied in the Chabot lab. Using an RNA version (RIP-chip) of the chromatin precipitation (ChIP) technique originally designed to assay DNA/protein interactions, he identified target RNAs bound by four hnRNP proteins. Borrowing from the transcription lexicography, he found that the respective hnRNP proteins often functioned together to either “silence” or “enhance” RNA processing.

“This study showed that factors of this protein family often co-regulate the same sets of genes,” says Blanchette, who continues this work at Stowers. “That suggests that the complexity of alternative splicing results from cooperative interactions among factors.”

He is now also analyzing mRNA-binding proteins known as exon-junction complex, or EJC, factors. Since the dawn of the molecular age, young gene jocks have been schooled in the maxim that “a spliced RNA is more expressible,” but until recently neither they nor most of their mentors had a clue why. It is now recognized that deposition of EJC factors on an RNA splice site is what enhances mRNA translation into protein. Blanchette’s lab is investigating mechanisms underlying how Drosophila EJC proteins regulate gene expression.

Blanchette was raised in the Québec town of Drummondville, 60 miles from Montréal. “I liked math and science in school and had all the toys that nerds have—a chemistry set and a little microscope,” he says. By the age of 10, Blanchette cut and welded steel alongside his father, who, although not formally trained as an engineer, was always building and engineering heavy machinery in his shop.

“I grew up around engines and my dad taught me how to put them together,” says Blanchette, calling the deconstruction of protein complexes “reverse-engineering”. “Now I just work on different kinds of machines.”