PMC

(A) Three DBA associated 5′UTR variants (c.-147_-146insGCCA, c.-147_-146insAGCC and c.-144_-141delTTTC), were generated by site directed mutagenesis from the pAcGFP-N1-382-S19-5′UTR clone (i.e. the “INTERMEDIATE” w.t. construct of 382 nt) using the Quick change II site directed mutagenesis (Stratagene) kit. (B) Western blot of total protein preparations isolated from transfected HEK293T and K562 cells performed as for . The constructs express each of the three structural 5′UTR variants. (C) Diagram showing the relative levels of RPS19 expressed from the three constructs in 293T and K562 cells, respectively. Quantification was made from Western blot analysis illustrated in (B). The expression from the w.t. “INTERMEDIATE” construct was used as a control and is set to 100.

(A) Sequences corresponding to three w.t. variants of the RPS19 mRNA including a 35 nt (SHORT), 382 nt (INTERMEDIATE) and 467 nt (LONG) 5′UTR, respectively, were introduced into the fluorescent reporter vector pAcGFP-N1 (Clontech) under the CMV promoter. The expressed fusion proteins consist of a full length RPS19 linked to green fluorescent protein (GFP). (B–C) RPS19 protein levels vary with different RPS19 5′UTR length. HEK293T and K562 cells were transfected with 5 µg of vector DNA from each of the three 5′UTR variants using Lipofectamine®2000 (Invitrogen). After 48 h, cells were assayed for expression of recombinant protein by fluorescence microscopy and stored at −20°C for further analysis by Western blot (B). (C) Diagram illustrating the relative expression of the three w.t. constructs in HEK293T and K562 cells, respectively. Quantification is based on Western blot analysis in (B) and the expression of RPS19-GFP fusion protein was normalized to β-actin.

(A) RPS19 5′UTR variants in testis and K562 cells. Schematic presentation of 39 different RPS19 5′UTRs identified of which 29 are yet undescribed. 5′RACE was performed with 1 µg of total RNA using the GeneRacer® kit (Invitrogen) according to manufacturer's recommendation. The RNA was treated with DNase I to clean samples from genomic DNA. The 5′RACE protocol selected full length G-capped mRNA and ruled out the possibility of partially degraded mRNA. PCR products were cloned into a TOPO-TA vector (Invitrogen) and 122 clones were picked randomly (83 from testis, 39 from K562 cells) and analyzed by bidirectional sequencing. The 5′UTR variants identified are indicated and aligned to the first exon of RPS19 from databases with a known maximum 5′UTR of 382 nt (bottom). (B) A schematic picture of the 5′ region of RPS19 cDNA (horizontal line) with relative positions of the start codon and the amplicons generated for quantification. Primers used to generate amplicons A, B and C for quantitative PCR are shown as arrows (sequences available upon request). (C) Tissue distribution of total RPS19 as determined by qPCR of amplicon A showing relative expression of RPS19 normalized to β-actin on a panel of primary human tissues and cell lines. Analyses were run in triplicates and the average is shown for each tissue. (D) Expression of the amplicons B and C representing longer variants of 5′UTR as determined by qPCR and expressed as a percentage of total RPS19 expression determined by amplicon A shown in (C).