To investigate the effect of NIP-2-H27A-N3 on the poly(A) tails of cellular mRNA, we utilized a commercially available poly(A) tail assay that relies on G/I tailing. In this assay, RNA is extracted from treated cells followed by the addition of alternating guanosine and inosine residues using a yeast poly(A) polymerase to the 3'-ends of poly(A)-containing RNA (supplemental Fig. ). The G/I tailed RNA is then converted to cDNA by reverse transcription using the newly added G/I tails as the priming sites. Next, the cDNA is analyzed via RT-PCR using two sets of primers. The first set has a forward primer selective for the gene of choice and a universal reverse primer which anneals to the junction between the poly(A) tail and the G/I tract. The second set of primers contains the same gene specific forward primer while the reverse primer anneals just before the poly(A) tail in the 3' UTR. After PCR amplification, both products are separated on an agarose gel and the difference in length correlates to the length of the poly(A) tail. HeLa cells were treated with the peptides for 24 hours followed by a treatment with actinomycin D to halt new RNA synthesis and subsequent RNA extraction after 1.5 hours. To select suitable genes for analysis we used the mRNA half-life dataset generated by Poetz et al. based on RNAseq analyses in the presence of an RNF219 mutant. This mutant protein is able to bind NOT9 and inhibit deadenylation by the CCR4-NOT complex but has no E3 ligase activity anymore and is therefore expected to have a similar effect as NIP-2. Two genes (RHOB and NR4A1) that had suitable expression levels in HeLa cells were selected and their poly(A) tails were analyzed in the absence or presence of NIP-2-H27A-N3. For RHOB, a significant decrease in the poly(A) tail is observed after new RNA synthesis is halted (compare 0 hours to 1.5 hours) (Fig. , supplemental Fig. ). When the cells are treated with compound, this decrease in the maximum poly(A) tail length was reversed. For NR4A1 the decrease in length between the DMSO treated 0 hour and 1.5 hours analyses is not significant but a strong increase in poly(A) tail length is observed in the compound treated cells (Fig. , supplemental Fig. ). For RHOB the poly(A) tail length increased with 13.1 ± 2.5 nucleotides while for NR4A1 the increase was 15.9 ± 4.5 nucleotides consistent with the findings of Poetz et al. that NOT9 inhibition stabilizes these mRNAs.