Post-transcriptional regulation of Ribosome Biogenesis in Larp1-deficient cells

Ribosome biogenesis is a critical aspect of cell differentiation. Ribosome synthesis has been previously reported to be regulated at the transcriptional and post-transcriptional levels. Poly(A) tail-length processing is a hallmark of post-transcriptional regulation associated with different steps of transcript metabolism. Here we monitor the contribution of the RNA-binding protein Larp1 in shaping the poly(A) tail profile of undifferentiated P19 cells. We found that Larp1 prevents the widespread shortening of poly(A) tails below 30 nucleotides and confers additional protection to transcripts containing a 5' terminal oligopyrimidine (TOP) motif, such as those encoding for ribosomal proteins. To knock down Larp1, undifferentiated P19 cells seeded in 6-well plates were transfected with 50 nanomoles of the Larp1 siRNA (Dharmacon, J-063706-09-0010) using Lipofectamine 3000 (Invitrogen) according to the manufacturer's instructions. A pool of scrambled siRNAs (Dharmacon, D-001810-10-20) with no significant homology to the human, mouse, or rat genome was used as a negative control. After transfection, cells were incubated at 37°C for 24 hours. To study the long-term effect at 48 hours, an additional transfection using the same conditions was performed 24 hours after the initial knock-down. RNA was collected, and Direct RNA seq libraries were prepared in duplicates or triplicates for each timepoint and run on a Nanopore GridIon device. Reads were mapped to the mouse transcriptome, and their poly(A) tail lengths were determined. This information was then used to monitor the impact of Larp1 knock-down on poly(A) tail length.