Post-Transcriptional Modifications of mRNA: 5’cap – a modified guanine. Critical for mRNA recognition and proper attachment to the ribosome. 3’poly-A tail – a long string of adenine nucleotides. Promotes export from the nucleus. Protects the mRNA transcript from degradation. Both cap and tail regulate mRNA splicing, transport, translation, and stability. mRNA

During translation mRNA is thought interact with several translation- initiation factors and the poly(A)-binding protein (PABP). The interaction causes circularization of the transcript and protects the 5' and 3’ ends from being attacked by deadenylase and decapping enzymes. Poly A Protection (Peltz et al, 2001)

Evidence for PolyA Tail Length Control of Gene Regulation The poly(A) tail is necessary for full miRNA-mediated repression of mRNA translation. (Humphreys et al, 2005). Under-adenylated maternal mRNAs are “stock-piled” in Xenopus oocytes. Via the action of cytoplasmic polyadenylation elements at oocyte maturation, the mRNAs become readenylated. This readenylation allows for active translation of the maternal mRNA. Phenotypes of yeast mutant for core deadenylase subunits implicate these enzymes as having important roles in controlling the cell cycle. (Woolstencroft et al, 2006; Pan et al, 2006).

Analysis of poly(A) tail length – 1.PAT analysis (PCR polyA test) – allows for the determination of polyA tail length of a specific mRNA species using RT-PCR. drawback – requiring transcript specific primers allows analysis of only one mRNA polyA tail at a time. 2.PASTA analysis (polyadenylation state array) – allows for the categorization of poly(A) tails as PASTA short or PASTA long. combines separation of cellular mRNA on polyU beads with microarray analysis. drawback – categories of “short” and “long” are broad and may fail to identify small changes in polyA tail length during mRNA regulation. We lack a method of determining polyA tail lengths for the entire transcriptome.

Step 1: Ligate cy-3 labeled oligo onto the 3’ end of total RNA.

Step 2: Hybridize to microarray. Step 3: Add the RT primers (sequence is complementary to the oligonucleotide).

Step 4: Reverse transcribe (on the microarray) using cy-5 labeled dTTP. Step 5: Measure fluorescence. Quantify the number of transcripts present. Use this value to normalize and assess poly(A) tail length.

Enzymatic Reactions on the Microarray Chip - Martha Bulyk et al have demonstrated the ability to perform second strand synthesis on an Affymetrix chip: Constant Priming Sequence Primer annealing confirmed with fluorescein-labeled primer. Enzymatic extension confirmed by two methods – 1)Fluorescein-labeled dATP and unlabeled dNTPs showed higher signal intensity corresponding to a greater proportion of adenine in the second strand. 2)3’ end labeling using fluorescein labeled ddNTP and terminal transferase. Only the 3’ end of the newly synthesized strand was available. (Bulyk et al, 1999) 5’ 3’

Step 1: Ligate cy-3 labeled oligo onto the 5’ end of total RNA.

T4 RNA ligase, is the product of gene 63 of the T4 phage. It catalyzes the ATP-dependent ligation of the 5’-end of ss DNA or RNA to the 3’-hydroxyl termini of DNA or RNA. Our goal - to investigate the efficiency of using this enzyme to ligate our oligo onto the end of total yeast RNA. T4 RNA Ligase

GAATCTCGGTCTCGAT-AAAAAAAA-rCrGrUrArCrA 1. 1 st strand synthesis - GAATCTCGGTCTTTCTGAT-AAAAAAAA-rCrGrUrArCrA 5’ 3’ dGdCdAdTdGdT 2. PCR using gene specific primer - dGdCdAdTdGdT Assessing ligation using RT-PCR -

Testing Fractionation with Synthetic RNA Markers A (20) A (60) A (40) 300 nt 600 nt 900 nt

Poly(A) Fractionation Technique to Separate RNAs Based on Their Tail Lengths AAAA Biotin oligo-dT + high salt solution Streptavidin coated beads TTTT B AAAAAAAAA TTTT B B AAAAAAAAA AAAA Wash Unbound RNA AAAAAAAAA TTTT B B S AAAA TTTT B Adapted from Meijer et al. 2007

Poly(A) fractionation continued AAAA AAAAAAAAA 1. Medium salt solution 2. Low salt solution AAAAAAAAA TTTT B B S TTTT B AAAA Adapted from Meijer et al Gehe GeneChip

Assessing ligation using DIG system - Digoxigenin-labeled nucleotide – Detected with an antibody conjugated to the enzyme alkaline phosphatase, which catalyzes a color reaction. Background and non-specific binding are typically low. Used to probe northern/southern blots.

5’-rCrGrUrArCrA + (1) 3’ end labeling of oligo with DIG - Terminal Transferase

AAAAAAAAA 5’-rCrGrUrArCrA AAAAAAAAA--rCrGrUrArCrA (2) Ligation of DIG labeled oligo to synthetic RNA - + Synthetic RNA: 320nt 640nt 960nt T4 Ligase

(3) Separate ligation products on a 2% agarose, 0.66M formaldehyde denaturing gel No ligation control DIG labeled oligo RNA ladder

LaneSample 1RNA Ladder 2320RNA+DIGoligo 3640RNA+DIGoligo 4960RNA+DIGoligo 5320RNA+DIGoligo 6640RNA+DIGoligo 7960RNA+DIGoligo 8320RNA 9640RNA 10960RNA

(4) Upward alkaline transfer of RNA to positively charged nylon membrane - (Brown, 2001)

LaneSample 1RNA Ladder 2320RNA+DIGoligo 3640RNA+DIGoligo 4960RNA+DIGoligo 5320RNA+DIGoligo 6640RNA+DIGoligo 7960RNA+DIGoligo 8320RNA 9640RNA 10960RNA

Phosphatase NBT/BCIP No DIG control DIG labeled oligo Unligated DIG oligo (5) Detection of DIG label by enzyme immunoassay -

LaneSample 1RNA Ladder 2320RNA+DIGoligo 3640RNA+DIGoligo 4960RNA+DIGoligo 5320RNA+DIGoligo 6640RNA+DIGoligo 7960RNA+DIGoligo 8320RNA 9640RNA 10960RNA

Future Direction / Problem Solving - End label the oligo with biotin? <--- may run into the same problems as with DIG. Hijack the luciferase assay used in pyrosequencing to detect the inorganic phosphate released upon ligation. Continue to optimize the system to allow for RT-PCR detection of the ligated products. Use radioactive labeling of the oligo to visualize the ligation product in the gel.