Vectors for expression of h9/14, h9/14A5, h9-3A/14 were described in (1,2). Plasmids encoding the eukaryotic translation initiation factors eIF1, 1A, 4A, 4B, 4G, 5, 5B and Escherichia coli Met-tRNA synthetase were described in (3). pGL3R-βglo, pGL3R-βact, pGL3R-L1, pGL3R-HCV, pGL3R-EMCV, pGl3R-CrPV were the kind gift of Dr. Sergey Dmitriev (4). PCR fragments amplified from these constructs and containing the Firefly luciferase ORF fused to 5ʼ-UTRs of β-globin, β-actin, LINE-1, HCV IRES or CrPV IRES under the control of SP6 promoter was inserted to pUC18 plasmid digested by HindIII and EcoRI to generate pUCβglo, pUCβact and pUCL1, and by PstI and EcoRI to generate pUCHCV and pUCCrPV. Vectors for in vivo expression of monocistronic mRNAs containing Firefly luciferase ORF fused to 5ʼ-UTRs of β-globin, β-actin and LINE-1 were prepared by insertion of PCR fragment containing respective 5ʼ-UTRs to pGL3R-EMCV digested by AflII and NcoI to remove upstream Renilla luciferase ORF and EMCV IRES. Plasmid pCrPV-VHLM was constructed by insertion of T7 promoter, CrPV IRES nucleotides 6028 to 6216 followed by a short ORF encoding Val-His-Leu-Met tripeptide, UAA stop codon and 100 nt random sequence taken from β-glucuronidase (GUS) gene, into the pUC18 vector. Plasmids pscAluYb8, pscAluYf2, pscAluYj4 were obtained from the plasmids pscAlu/α-feto and pSscAlu/LDL (1) using quick change protocol. pSAlu110 and pSAlu151 contained the Alu portion of 7SL RNA gene (nucleotides 1-74; 271-299 and 1-99; 251-299, respectively) with a closing loop GATT following position 74 or 99, preceded by the T7 promoter. [ 32 P]-MVHL-stop mRNA synthesis [ 32 P]-UTP was added to the transcription reaction. Preprolactin and MVHL-stop mRNAs were synthesized with SP6 RNA polymerase from plasmids pSP-BP4 (8) and MVHL-stop (9) linearized with EcoRI and XhoI. Capped and uncapped RNAs encoding Firefly luciferase fused to various 5ʼ-UTRs were synthesized with SP6 RNA polymerase from plasmids pUCβglo, pUCβact, pUCL1, pUCCHV and pUCCrPV linearized with EcoRI. HCV-NSʼ and CrPV-VHLM mRNAs were synthesized with T7 RNA polymerase from plasmids pXL.HCV(40-373).NSʼ (10) and pCrPV-VHLM linearized with EcoRI and SspI, respectively. The mRNAs were purified by LiCl precipitation, G50 sepharose chromatography, ethanol precipitation, and resuspended in water. Non-coding AluY A , scAluY A , scAluY L , and 4.5S RNAs were synthesized with T7 RNA polymerase from plasmids pPAlu, pPscAlu/a feto, pSscAlu/LDL and pS4.5S (1) linearized with SspI, SpeI, SpeI and Andreev, D.E., Dmitriev, S.E., Terenin, I.M., Prassolov, V.S., Merrick, W.C. and Shatsky, I.N. (2009) Differential contribution of the m7G-cap to the 5' end-dependent translation initiation of mammalian mRNAs. Nucleic Acids Res., 37, 6135-6147. 5. Dewannieux, M., Esnault, C. and Heidmann, T. (2003) LINE-mediated retrotransposition of marked Alu sequences. Nat. Genet., 35, 41-48. 6. Gurevich, V.V., Pokrovskaya, I.D., Obukhova, T.A. and Zozulya, S.A. (1991) Preparative in vitro mRNA synthesis using SP6 and T7 RNA polymerases. Anal. Biochem., 195, 207-213. 7. Milligan, J.F. and Uhlenbeck, O.C. (1989) Synthesis of small RNAs using T7 RNA polymerase. Methods Enzymol., 180, 51-62. 8. Siegel, V. and Walter, P. (1988) Each of the activities of signal recognition particle (SRP) is contained within a distinct domain: analysis of biochemical mutants of SRP. Cell, 52, 39-49. 9. Alkalaeva, E.Z., Pisarev, A.V., Frolova, L.Y., Kisselev, L.L. and Pestova, T.V. (2006) In vitro reconstitution of eukaryotic translation reveals cooperativity between release factors eRF1 and eRF3. Cell, 125, 1125-1136.
Output: SUPPLEMENTARY DATA Alu RNA regulates the cellular pool of active ribosomes by targeted delivery of SRP9/14 to 40S subunits