extracting exon0 isolating a mec-8 isoform

04/20/23 1

Extracting Exon0Isolating a mec-8 Isoform

Sol Katzman

Sol Katzman 04/20/23 2

Zahler lab -- C. elegans“Alt splicing, all the time” Where’s the binf? Uncoordinated worms Mixing colorless liquids

from http://elegans.swmed.edu/

Sol Katzman 04/20/23 3

unc-52 exons 17,18 optionalmediated by mec-8

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mec-8 four exons +C43002

Sol Katzman 04/20/23 5

mec-8 zoom out to see Exon0A 6500bp intron in C. elegans?

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mec-8 (Wormbase view)

Sol Katzman 04/20/23 7

5’ RACE procedures5’

mRNA3’

gsp1

reverse transcribe

3’cDNA

5’

purify

3’cDNA

5’

tail

3’cDNA

5’

PCR

CCCCCAAPgsp2

5’dsDNA

3’ AAPgsp2

Sol Katzman 04/20/23 8

trans-spliced isoforms and primers.known and unknownshould be at least 85bp difference

exon4 exon3 exon2 exon1

exon4 exon3 exon2 exon1 exon0

SL1

SL1?

SL1

SL1

AAP

AAP

3’

3’ 5’

5’tailed

tailed

ex0for

ex01revgsp2

gsp2

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5’ RACE gel using AAP and SL1 forward primers

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TOPO-TA cloning

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TOPO clone vector

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plasmid digestion after cloning

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First clones: exon1 and 2, plus SL1

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trans-spliced isoforms and primers.try exon0forward (and other RNA)

exon4 exon3 exon2 exon1

exon4 exon3 exon2 exon1 exon0

SL1

SL1?

SL1

SL1

AAP

AAP

3’

3’ 5’

5’tailed

tailed

ex0for

ex01revgsp2

gsp2

Sol Katzman 04/20/23 15

Exon0For primer on First Strand cDNA3 RNA samples: embryo, L4, mixed-stageFound something!

Sol Katzman 04/20/23 16

5’ RACE procedures5’

mRNA3’

gsp1

reverse transcribe

3’cDNA

5’

purify

3’cDNA

5’

tail

3’cDNA

5’

PCR

CCCCCAAPgsp2

5’dsDNA

3’ AAPgsp2

Sol Katzman 04/20/23 17

Exon0For primer: fading through 5’RACE procedure steps. (embryo RNA only)

Sol Katzman 04/20/23 18

trans-spliced isoforms and primers.try exon01reverse primer

exon4 exon3 exon2 exon1

exon4 exon3 exon2 exon1 exon0

SL1

SL1?

SL1

SL1

AAP

AAP

3’

3’ 5’

5’tailed

tailed

ex0for

ex01revgsp2

gsp2

Sol Katzman 04/20/23 19

SL1 forward - Exon01Rev primerson purified cDNA. Double band at 200bp

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Clone and PCR sequences – BLAT.2 versions of exon0. SL1 leaders.Where’s the ORF?

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To Do

find promoter and bash it investigate conservation regions in intron use the third worm -- C. remanei consider uORFs

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Acknowledgements

Al Zahler Sam Gu Jen Kabat

Val, Brian, Mary Ann, Poojitha, Joy, Sergio U.C. Regents