The research was conducted over the course of the last year and a half by teams from the laboratories headed by Pamela J. Green, Crawford H. Greenewalt Endowed Chair in Plant Molecular Biology, a joint appointment in the Department of Plant and Soil Sciences and the College of Marine Studies, and Blake C. Meyers, assistant professor of plant and soil sciences in the College of Agriculture and Natural Resources.
To identify the small RNAs, the scientists used the transcriptional profiling technology called Massively Parallel Signature Sequencing (MPSS), which was developed by Solexa Inc. of Hayward, Calif.
Green and Meyers pioneered the application of MPSS to small RNAs in collaboration with scientists at Solexa.
Green said that small RNAs are "one of most important discoveries in biotechnology in the last 10 years" because they play an important role in regulating genes in both plants and animals.
Deficiencies in small RNA production can have a profound effect on development, and small RNAs have been associated with other important biological processes, such as responses to stress.
Determining the sequence of the small RNAs of an organism is critical for understanding their overall impact and individual biological roles, Meyers said.
Although several thousand small RNAs have been identified from diverse plant and animal systems, these sequences were identified using older technologies that do not sequence deeply enough to characterize these molecules on a genome-wide scale. Quantitative information about the abundance and regulation of the majority of small RNAs also has been lacking.
With funding from the National Science Foundation, the laboratories of Green and Meyers, which are housed at the Delaware Biotechnology Institute, overcame these obstacles to make a breakthrough in the study of small RNAs from Arabidopsis.
Prior to their work, scientists worldwide in a painstakingly slow and labor-intensive process had documented about 6,000 small RNAs from the plant.
Meyers had been sequencing RNAs in rice and Arabidopsis using MPSS when Green approached him about the possibility of sequencing small RNAs using the MPSS technology.
"We knew MPSS could work in sequencing small RNAs but we were not sure how interesting the outcome would be," Meyers said. "But, as soon as we received the first complete data set, we quickly saw that it was far richer and more complex than anyone had previously generated for this type of molecule."
As the project progressed, the laboratories sequenced about 2.2 million small RNAs from the seedlings and flowers of the plant and identified more than 75,000 different small RNA sequences.
"Not only does MPSS provide very deep coverage of small RNAs, but it also provides quantitative information," Green said, adding that "this allowed many highly regulated small RNAs to be identified."
In addition to the sheer number of sequences identified, Meyers said "the biggest surprise in the findings is the diversity." He said that their data indicated that the regions of the chromosomes where people had speculated there was not much transcriptional activity turned out to be sites of tremendous amounts of small RNA activity.
Green said the implications of their findings would have a vital impact on future research by both their laboratories and those at other institutions. "What we found is just the beginning because the ramifications go way beyond that," she said. "Much of the future excitement will result from different laboratories testing new ideas after looking at the data for their favorite genes or chr omosomal regions."She explained that the field of study is booming because small RNAs are important in their regulation of large numbers of genes in complex biological systems. One small RNA can regulate multiple genes, and researchers believe that more than10 percent of human genes are being regulated by small RNAs.
Small RNAs are characterized by a length of approximately 21 to 24 nucleotides. The biological activity of small RNAs was first described about 12 years ago, but the most substantial advances in this field have 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.
Science is a prestigious journal published by the American Association for the Advancement of Science. The paper's first and second authors are Cheng Lu, a molecular biology postdoctoral researcher, and Shivakundan Singh Tej, a computer science student. Also among the authors, in addition to Green and Meyers, are Shujun Luo and Christian D. Haudenschild of Solexa.
"By working closely together, and with industrial collaborators," Green said, "the students and postdocs crossed the boundaries of their conventional training. This emulates the goals of UD and DBI for interdisciplinary research, education and economic development."
In addition to funding by NSF, the laboratories have received grants from DBI and the U.S. Department of Agriculture to continue their work on small RNA analysis.