ncRNAStructural

1. rRNA2. tRNA3. snRNA4. snoRNA5. cleavage: Rnases P & MRP, U3, snR30, etc

Regulatory1. Small• siRNA• miRNA

2. Long• Activator• Enhancer• silencing

Other ncRNA?Incredible diversity of

functions!• Epigenetic• Directly regulating

transcription• Post-transcriptional

regulationSome are made by Pol

II, others by Pol III

Our ncRNA1. Extracted total RNA, separated out and sequenced 50-

300 nt

Our ncRNA1. Extracted total RNA, separated out and sequenced 50-

300 nt2. BLAST to find relatives (and discard tRNA, etc)

Our ncRNA1. Extracted total RNA, separated out and sequenced 50-

300 nt2. BLAST to find relatives (and discard tRNA, etc)3. T-DNA express to find mutants

Our ncRNA1. Extracted total RNA, separated out and sequenced 50-

300 nt2. BLAST to find relatives (and discard tRNA, etc)3. T-DNA express to find genes that weren’t near protein-

coding genes

Our ncRNA1. Extracted total RNA, separated out and sequenced 50-

300 nt2. BLAST to find relatives (and discard tRNA, etc)3. T-DNA express to find genes that weren’t near protein-

coding genes4. 33 Hits: order seeds

Studying your ncRNA1.Does it resemble anything known?• Remember that many ncRNA bind known targets

Studying your ncRNA1.Does it resemble anything known?• Remember that many ncRNA bind known targets

2.Can it adopt a stable structure?

Studying your ncRNA1.Does it resemble anything known?• Remember that many ncRNA bind known targets

2.Can it adopt a stable structure?3.Has its expression been detected by other means?• MPSS, microarrays, etc

Studying your ncRNA1.Design primers to identify homozygous plants• Seeds are supplied as heteroygotes to avoid obligate

lethals

Studying your ncRNA1.Design primers to identify homozygous plants• Seeds are supplied as heteroygotes to avoid obligate

lethals2.Design primers to detect your ncRNA3.Grow seeds of your line, look for plants that look weird and collect their seeds.

Primer/probe design• Stem-loop primers for short RNAs where only have enough info for one primer