Genome annotation by high-throughput 5’ RNA end determination

1. Genome annotation by high-throughput 5’ RNA end determination Byung Joon Hwang, Hans-Michael Müller, and Paul W. Sternberg

2. Paul W. Sternberg • B.A. degree from Hampshire College • Ph.D. degree from M. I. T. under Robert Horvitz • Postdoctoral research with Ira Herskowitz at the U. C., San Francisco • Professor of Biology at the California Institute of Technology and Adjunct Professor of Cell and Neurobiology at the University of Southern California School of Medicine, Los Angeles

3. Sternberg Lab • Byung Joon Hwang – Post Doc • Hans-Michael Müller - Wormbase

4. Trans-spliced Exon Coupled RNA End Determination (TEC-RED) • Identifies 5 ’ ends of expressed genes • Can distinguish coding regions from regulatory regions • Useful for identifying genes with alternatively spliced 5 ’ ends. • Developed in nematodes, but can work for any organism which use a spliced leader sequence (Sarcomastigophora, cndarians, nematodes, acoelomate flatworms and ascidians).

5. Trans-splicing • 70% of mRNAs have one of two splice-leader sequences (SL1, SL2) trans-spliced onto the 5’ end • Spliced-leader sequences are transcribed independently as snRNA’s • Trans-splicing with splice-leader sequences produces single-gene mRNAs

7. TEC-RED

9. Sequential Concentration • Eliminates the large-scale PCR reactions and gel purification of small oligonucleotides steps found in SAGE protocols. • Since the 5’ tags are directionally concentrated, the 5’ end of the 5’ tags are found next to the first anchor RE cut site.

10. DNA Sequence Analysis

11. Data • 13 525 5’ tags (9 401 with SL1 and 4 124 for SL2), obtained from 800 sequencing reactions, were matched to the genome • Represents 2 159 different sequences, 1 639 for SL1 and 520 for SL2 • 90% of tags corresponded to unique sites • Of the remaining 10%, 90% matched 2 or 3 sites

12. Cont. • To remove false positives they analyzed the just 5’ of the 5’ tag sites for a conserved splice acceptor consensus sequence • 93% of the 5’ tag sequences remained true positives

13. …. • 75% of tag sequences matched know 5’ end in WS100 • 99 new genes identified • 32 previously unknown operons identified

14. Conclusion • Method for annotation of 5’ end of genes. • This protocol works for only organisms with a splice leader sequence • Sequential concentration method applied to SAGE protocols

15. “The United States brags about its political system, but the President says one thing during the election, something else when he takes office, something else at midterm and something else when he leaves.” -Deng Xiaoping