ncRNA Structural

1. rRNA 2. tRNA 3. snRNA 4. snoRNA 5. cleavage: Rnases P & MRP, U3, snR30, etc Regulatory 1. Small

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siRNA

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miRNA 2. Long

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Activator

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Enhancer silencing

Other ncRNA?

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Incredible diversity of

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functions!

Epigenetic Directly regulating transcription Post-transcriptional regulation Some are made by Pol II, others by Pol III

Our ncRNA 1. Extracted total RNA, separated out and sequenced 50 300 nt

Our ncRNA 1. Extracted total RNA, separated out and sequenced 50 300 nt 2. BLAST to find relatives (and discard tRNA, etc)

Our ncRNA 1. Extracted total RNA, separated out and sequenced 50 300 nt 2. BLAST to find relatives (and discard tRNA, etc) 3. T-DNA express to find mutants

Our ncRNA 1. Extracted total RNA, separated out and sequenced 50 300 nt 2. BLAST to find relatives (and discard tRNA, etc) 3. T-DNA express to find genes that weren’t near protein coding genes

Our ncRNA 1. Extracted total RNA, separated out and sequenced 50 300 nt 2. BLAST to find relatives (and discard tRNA, etc) 3. T-DNA express to find genes that weren’t near protein coding genes 4. 33 Hits: order seeds

Studying your ncRNA

1. Does it resemble anything known?

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Remember that many ncRNA bind known targets

Studying your ncRNA

1. Does it resemble anything known?

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Remember that many ncRNA bind known targets 2. Can it adopt a stable structure?

Studying your ncRNA

1. Does it resemble anything known?

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Remember that many ncRNA bind known targets 2. Can it adopt a stable structure?

3. Has its expression been detected by other means?

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MPSS, microarrays, etc

Studying your ncRNA

1. Design primers to identify homozygous plants

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Seeds are supplied as heteroygotes to avoid obligate lethals

Studying your ncRNA

1. Design primers to identify homozygous plants

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Seeds are supplied as heteroygotes to avoid obligate lethals 2. Design primers to detect your ncRNA 3. Grow seeds of your line, look for plants that look weird and collect their seeds.

Primer/probe design

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Stem-loop primers for short RNAs where only have enough info for one primer