Microarray analysis of Protein Bound RNA transcripts

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Microarray analysis of Protein Bound RNA
transcripts

• Richard Rogers
• Research Scientist
• Aitchison Lab

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Overview

• Background on Agilent arrays
• Protocol we use to label RNA
• Data analysis of protein bound RNA transcripts
• She2--RNA-binding protein that binds specific
  mRNAs part of the mRNA localization machinery
  that restricts accumulation of certain proteins
  to the bud
• Mex67--Poly(A)RNA binding protein involved in
  nuclear mRNA export
• Nop15--Constituent of 66S pre-ribosomal
  particles, involved in 60S ribosomal subunit
  biogenesis
• Oligo dT primer vs. Random primer
• Summary

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Agilent Technology

• in situ oligonucleotide probe synthesis
• No physical masks or printing plates allows for
  flexible content in principle each array can
  have a different design
• Traditionally expensive, but recent density
  increases make Agilent more cost competitive,
  especially with multiple arrays per slide
• 2-color (or 1-color)

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Agilent Yeast Arrays

• ORF 8 x 15K arrays (1 x 6,000 probes)
• 750/slide gt 94/array
• Must hybridize all 8 arrays at once
• Tiling 4x44K
• 656/slide gt 164/array
• 290bp resolution
• Tiling 244K
• 431/slide/array
• 50bp resolution
• Extremely flexible for custom designs
• Web site for designing arrays
• No setup costs other microarray vendors charge
  1,000s to 10,000s

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Agilent Labeling options

• Agilents kit 160 reagents/array (2 colors)
• Start with 50ng 5ug total RNA
• Standardized, it works
• IVT amplification may be overkill
• Numerous 3rd party labeling reagents and kits
• Cheaper
• Cy-dyes, Alexa dyes, others?
• cDNA synthesis incorporating labeled nucleotides
• or
• cDNA synthesis with modified nucleotides for
  subsequent conjugation to fluorescent dyes

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Amplification and Labeling of RNA
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She2 associated transcripts vs. Total RNA
Scatter Plot
Parallel Plot
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Enriched She2 transcripts

• We observed 400 transcripts significantly
  enriched in our She2 data
• High correlation with previous studies
• Enriched transcripts are associated with the GO
  component annotation of cellular bud (1.50E-08)
• We identified significantly more transcripts than
  previous studies
• Suggesting that She2 associated transcipts may be
  involved in localizing transcripts to other
  organelles (determined by GO annotation analysis)

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Mex67 associated transcripts vs. Total RNA
Scatter Plot
Parallel Plot
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Enriched Mex67 transcripts

• We identify 230 mRNA transcripts bound to Mex67
• Silver lab identified 1140 transcripts bound to
  Mex67
• There is a high degree of correlation between
  both data sets (30 are similar)
• the 30 in common are highly transcribed genes
  (average mRNA/hr 43.6)
• The difference in transcripts identified could be
  due to different cut-offs for significant data or
  may be an indication of the sensitivity and
  accuracy of the IP technique

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Nop15 is involved in rRNA processing
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Custom array for Nop15 contained rRNA probes

• 5S
• 5.8S
• 25S
• 18S
• ITS1 Internal transcribed spacer 1
• ITS2 Internal transcribed spacer 2
• ETS1 External transcribed spacer 1
• ETS2 External transcribed spacer 2
• NTS1 Non transcribed spacer 1
• NTS2 Non transcribed spacer 2

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Nop15 associated transcripts vs. Total RNA
rDNA probes are highlighted in red
Parallel Plot Oligo dT Primer
Parallel Plot Random Primer
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• rRNA can be polyadenylated
• Pap1p, the poly(A) polymerase, is responsible for
  adenylating the rRNAs
• For at least one rRNA type, the polyadenylation
  preferentially occurs on the precursor rather
  than the mature product
• polyadenylated rRNAs may reflect a
  quality-control mechanism of rRNA biogenesis

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Nop15 associated transcripts vs. Total RNA
ITS probes are highlighted in red
Parallel Plot Oligo dT Primer
Parallel Plot Random Primer
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Nop15 enriched transcripts

• Marlene has previously reported that Nop15 is
  required for normal pre-rRNA processing,
  specifically 5.8S and 25S
• In the Oligo dT amplified Nop15 sample there is a
  significant enrichment of Its2, 5.8S, and 25S
• The ratios for the random primed sample vs. the
  oligo dT sample are similar

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Summary

• Agilent Technoligies has made the price of
  expression arrays and chip-on-chip arrays
  reasonable
• Our She2 and Mex67 data agree with previously
  published data
• The quick IP technique may provide a better
  understanding of dynamic interacting proteins and
  RNA trancripts
• Our Nop15 data shows that we can pinpoint the
  step of rRNA processing for a protein
• The Nop15 data also suggests that polyadenylation
  of rRNA may play bigger role rRNA processing than
  previously thought although further studies need
  to be done

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