Luciferase constructs

To obtain Mbnl1_5΄UTR2_Luc2 Luc vector, pmirGLO (Promega) was digested with NheI and XbaI, ligated and then annealed oligos (F10/R10) where cloned into a HindIII site in front of Luc2. +e1_Luc and –e1_Luc constructs were obtained in a few sequential steps. First, Mbnl1 fragments were amplified with F11/R11 primers from C2C12 cDNA, and then the 5΄UTR products were re-amplified with F12/R12.1 or F12/R12.2 primers and cloned into Luc vector with NheI and XhoI. Such obtained +e1_Luc and –e1_Luc constructs contained 132 nucleotides of e5΄UTR2 and the full 5΄UTR sequence of either e1 (875 nucleotides) or e2 (102 nucleotides) between the promoter and Luc2, respectively. The constructs retained short vector/multiple cloning site sequences downstream of the promoter and upstream of Luc2.

Luciferase Mbnl1_e1_5΄UTR2_Luc2 constructs were generated based on pGL4.51 (Promega) and pmirGLO vectors. Mbnl1 fragment was PCR-amplified using F13/R11 primers on a template of RNA obtained from mouse NIH/3T3 cells, and then a shorter fragment was generated using F14/R2 primers. The sequence contained 65 nucleotides of e5΄UTR2 and 863 nucleotides of e1. One ATG codon in e5΄UTR2 was mutated [G to T; chr3: 60501374 (mm10)] using the forward primer to prevent expression of a shorter protein that could delay the translation of luciferase. Obtained Mbnl1 fragment was digested with HindIII and cloned into HindIII digested pGL4.51 vector. To obtain Ctrl construct, a PCR fragment was amplified with F15/R15 primers, digested with MluI and XhoI, and cloned into MluI and XhoI-digested pmirGLO. Δ1 and Δ2 were obtained by digestion of Ctrl with SmaI and XbaI. The ends were blunted with the Klenow fragment (Thermo Scientific) prior to the ligation. Deleted sequences in Δ1 and Δ2 constructs are listed in Supplementary Table S2. An empty vector lacking Mbnl1 (Emp) was generated by removing the Mbnl1 fragment from Ctrl vector with HindIII digestion.