Delaware Scientists Make Significant Advance In Study Of Small RNAs

Theresearch was conducted over the course of the last year and a half byteams from the laboratories headed by Pamela J. Green, Crawford H.Greenewalt Endowed Chair in Plant Molecular Biology, a jointappointment in the Department of Plant and Soil Sciences and theCollege of Marine Studies, and Blake C. Meyers, assistant professor ofplant and soil sciences in the College of Agriculture and NaturalResources.

To identify the small RNAs, the scientists used thetranscriptional profiling technology called Massively ParallelSignature Sequencing (MPSS), which was developed by Solexa Inc. ofHayward, Calif.

Science is a prestigious journal published by theAmerican Association for the Advancement of Science. The paper’s firstand second authors are Cheng Lu, a molecular biology postdoctoralresearcher, and Shivakundan Singh Tej, a computer science student. Alsoamong the authors, in addition to Green and Meyers, are Shujun Luo andChristian D. Haudenschild of Solexa.

Green and Meyers pioneered the application of MPSS to small RNAs in collaboration with scientists at Solexa.

Greensaid that small RNAs are “one of most important discoveries inbiotechnology in the last 10 years” because they play an important rolein regulating genes in both plants and animals.

Deficiencies insmall RNA production can have a profound effect on development, andsmall RNAs have been associated with other important biologicalprocesses, such as responses to stress.

Determining the sequenceof the small RNAs of an organism is critical for understanding theiroverall impact and individual biological roles, Meyers said.

Althoughseveral thousand small RNAs have been identified from diverse plant andanimal systems, these sequences were identified using oldertechnologies that do not sequence deeply enough to characterize thesemolecules on a genome-wide scale. Quantitative information about theabundance and regulation of the majority of small RNAs also has beenlacking.

With funding from the National Science Foundation, thelaboratories of Green and Meyers, which are housed at the DelawareBiotechnology Institute, overcame these obstacles to make abreakthrough in the study of small RNAs from Arabidopsis.

Priorto their work, scientists worldwide in a painstakingly slow andlabor-intensive process had documented about 6,000 small RNAs from theplant.

Meyers had been sequencing RNAs in rice and Arabidopsisusing MPSS when Green approached him about the possibility ofsequencing small RNAs using the MPSS technology.

“We knew MPSScould work in sequencing small RNAs but we were not sure howinteresting the outcome would be,” Meyers said. “But, as soon as wereceived the first complete data set, we quickly saw that it was farricher and more complex than anyone had previously generated for thistype of molecule.”

As the project progressed, the laboratoriessequenced about 2.2 million small RNAs from the seedlings and flowersof the plant and identified more than 75,000 different small RNAsequences.

“Not only does MPSS provide very deep coverage ofsmall RNAs, but it also provides quantitative information,” Green said,adding that “this allowed many highly regulated small RNAs to beidentified.”

In addition to the sheer number of sequencesidentified, Meyers said “the biggest surprise in the findings is thediversity.” He said that their data indicated that the regions of thechromosomes where people had speculated there was not muchtranscriptional activity turned out to be sites of tremendous amountsof small RNA activity.

Green said the implications of theirfindings would have a vital impact on future research by both theirlaboratories and those at other institutions. “What we found is justthe beginning because the ramifications go way beyond that,” she said.“Much of the future excitement will result from different laboratoriestesting new ideas after looking at the data for their favorite genes orchromosomal regions.”

She explained that the field of study isbooming because small RNAs are important in their regulation of largenumbers of genes in complex biological systems. One small RNA canregulate multiple genes, and researchers believe that more than 10percent of human genes are being regulated by small RNAs.

SmallRNAs are characterized by a length of approximately 21 to 24nucleotides. The biological activity of small RNAs was first describedabout 12 years ago, but the most substantial advances in this fieldhave only been made in the last six or seven years.

To assist other small RNA researchers, the UD team has created a user-friendly web site [http://mpss.udel.edu/at] available to scientists to examine any gene or region of an Arabidopsis chromosome for matches to small RNAs.

“Byworking closely together, and with industrial collaborators,” Greensaid, “the students and postdocs crossed the boundaries of theirconventional training. This emulates the goals of UD and DBI forinterdisciplinary research, education and economic development.”

Inaddition to funding by NSF, the laboratories have received grants fromDBI and the U.S. Department of Agriculture to continue their work onsmall RNA analysis.