Sequencing Primary MicroRNA Transcripts in Oryza Sativa

1. Sequencing Primary MicroRNA Transcripts in Oryza Sativa Summer 2006 Johnnie Walker Brenda Zosa Cindi Lewis Neena Agrawal Claflin University University of South Carolina

2. BackgroundDefinition of microRNA • Small, RNA molecules encoded in the genomes of plants and animals • highly conserved, ~21-mer RNAs regulate the expression of genes • Made as precursors • In animals primarily bind to the 3'-untranslated regions (3'-UTR) of specific mRNAsand block translation • In plants they bind anywhere on the transcript and generally cleave them

3. miRNA Processing and Gene Regulation • Dicer Like 1 processing of primary transcript in nucleus for plants • Stem-Loop structure • RISC incorporation • Cleaves target mRNA

4. Characteristics of Primary Transcripts • Made by RNA polymerase II • 5’ end cap • 3’ end Poly(A) Tail • Contains Pre-miRNA stem-loop sequence

5. Why Study miRNAs? • Recently discovered (2001) major mechanism of gene regulation in all eukaryotic organisms • Plays a crucial role in development • Important for response to stress Why Study miRNAs in Rice? • Practical applications such as: • Enhance/improve growth • Produce stronger plants • Improve yield, stress response • Improve sustainability in various climates

6. Objectives • Map the 5’ and 3’ ends of miRNA precursors to the O. sativa genome • Determine whether the miRNA precursors have introns • Determine whether the miRNA precursors are located in intergenic regions or in protein coding regions

7. Methods • Growing Rice • RNA Extraction • 5’Rapid Amplification of cDNA Ends (R.A.CE.) • 3’Rapid Amplification of cDNA Ends (R.A.C.E) • Primer Design • PCR • Cloning • Mapping

8. Growing Rice • Rice was grown in a growth chamber and Greenhouse 2 using a mix of soil and vermiculite. • Rice achieves optimal growth with short days and long nights to promote flowering.

9. Extraction of RNA • Homogenization: Grinding tissue in Trizol Reagent • Phase Separation: Incubation and separation by centrifuging • RNA precipitation: supernatant and isopropanol incubation • TRI-Reagent Cleanup: extraction and separation

10. 5’ RACE • Isolate only RNA that has a cap • Isolate total RNA from cell • Remove 5’-phosphate from all RNA that is not capped • Remove cap from remaining RNA • Add 5’ adapter from RACE kit to the 5’ ends of RNA • Create cDNA of modified RNA with a reverse transcriptase reaction to use in the PCR reactions

11. 3’ RACE • Add the 3’ adaptor to extracted RNA- Ligates to polyA tail only • Reverse Transcription reaction to make cDNA to be used in PCR

12. Primer Designing • Chose miRNA genes according to abundance of small RNAs • Gene specific primers were based on predicted pre-miRNA gene sequences

13. PCR Selective Amplification of Target Sequences • Denaturation • Annealing • Elongation

14. Cloning • PCR products were inserted into a plasmid vector from the Invitrogen TOPO TA Cloning Kit and transformed into Escherichia coli cells. • Plasmids from cells were sent to Northwoods DNA, Inc. for sequencing

15. Mapping of Sequences • Search for specific primer sequences and miRNA sequences in the sequences sent back • Once sequence has been found, input it into the BLAST website, remembering not to include the adapter sequence • Determine if sequence is on the correct chromosome, in the correct place • Check to see if there are any introns or adjacent protein-coding genes near the sequence

16. Results • 5’ Ends: • Neena • Johnnie • 3’ Ends: • Brenda • Cindi

17. Neena’s Results • 167g • Mapping Results: • Location – Ch 3: 3326651-3326854 (miRNA – 3326803-3326823, 152 bp from the 5’-end) • No introns • Located within a protein-coding region – expressed protein LOC_Os03g06640 • 397b • Mapping Results: • Location – Ch 2: 3280789-3280809 (miRNA – 3280866-3280886, 239 bp from the 5’-end) • No introns • Adjacent Genes • 5’: expressed protein LOC_Os02g06560 • 3’: CBL-interacting serine/threonine-protein kinase 15, putative, expressed LOC_Os02g06570 • 167h • Mapping Results: None Yet • 393b • Mapping Results: None Yet

18. Brenda’s Results • miRNA 397b 3’ End • 3279937-3280339-intron-3280619-3280826 • 609 bps long sequence • Intron 280 bps • ~929 bases down from end of miRNA

19. Johnnie’s Results • After analyzing sequences for miRNA gene 167c, PCR was found to have amplified the wrong sequence. • RACE kit inner primer was found to be complementary to a sequence downstream of the RACE kit adapter in the cDNA template used in PCR. MicroRNA genes 166d and 169c samples were sent off for sequencing.

20. Cindi’s Results • 169p: • Sample #7- Sequence length:243 nt. • Location: 7634787 -7635029, with no introns present • miRNA Precursor location:7634985-7635110 • miRNA location:7635100- 7635079, 313 nt.’s upstream from the 3’ end of cloned sequence • Two putative transposon proteins are located on either end at considerable distances • 167c: • Several ends found with primer pairs being unable to be located successfully in cloned sequences • 166d & 171 b: • Data yet to be received from sequencing

21. Acknowledgements • Dr. Oluwole Ariyo • Dr. Vicki Vance • Dr. Lewis Bowman • The Vance Lab • The Bowman Lab