PRPF19 mRNA encodes a small open reading frame that is important for viability of human cells

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Abstract

High-throughput ribosome profiling demonstrated the translation of thousands small open reading frames located in the 5′ untranslated regions of messenger RNAs (upstream ORFs). Upstream ORF can both perform a regulatory function by influencing the translation of the downstream main ORF, and encode a small functional protein or microprotein. In this work, we showed that the 5′ untranslated region of the PRPF19 mRNA encodes an upstream ORF that is translated in human cells. Inactivation of this upstream ORF reduces the viability of human cells.

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About the authors

N. M. Shepelev

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences; Lomonosov Moscow State University

Author for correspondence.
Email: mprubtsova@gmail.com

Department of Chemistry

Russian Federation, Moscow; Moscow

А. О. Kurochkina

Lomonosov Moscow State University

Email: mprubtsova@gmail.com

Department of Chemistry

Russian Federation, Moscow

О. А. Dontsova

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences; A. N. Belozersky Institute of Physico-Chemical BiologyLomonosov Moscow State University; Skolkovo Institute of Science and Technology

Email: mprubtsova@gmail.com

Academician, Department of Chemistry, Center for Molecular and Cellular Biology

Russian Federation, Moscow; Moscow; Moscow; Moscow

M. P. Rubtsova

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences; Lomonosov Moscow State University

Email: mprubtsova@gmail.com

Department of Chemistry

Russian Federation, Moscow; Moscow

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Supplementary files

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2. Fig. 1. Ribosomal profiling of the main mRNA isoform of the human PRPF19 gene (ENST00000227524) according to the Ensemble database, displayed in the Trips-Viz transcriptome browser. In the diagram below, three possible reading frames and their corresponding profiles on the graph are marked in different colors. On the reading frame diagram, stop codons (UGA, UAA, UAG) are marked in black, and start codons (AUG) are marked in white.

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3. Fig. 2. Profiling of initiating ribosomes of the main mRNA isoform of the human PRPF19 gene (ENST00000227524) according to the Ensemble database, displayed in the Trips-Viz transcriptome browser. In the diagram, three possible reading frames and their corresponding profiles on the graph are marked in different colors. In the diagram below, stop codons (UGA, UAA, UAG) are marked in black, start codons (AUG) are marked in white.

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4. Fig. 3. Confirmation of translation of the upstream ORF of the PRPF19 gene using a reporter construct: scheme of the reporter construct. Part of the PRPF19 ORF is connected to the EGFP ORF. vORS – overlying ORS (A); measurement of HiBiT-mediated luciferase activity in cell lysates after transfection with reporter constructs. Means and standard deviations for 4 independent transfections are shown. RLU – relative light units; *** – p-value < 0.001 (B).

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5. Fig. 4. Analysis of the conservation of the upstream ORF of the PRPF19 gene among mammals: alignment of the upstream ORF of the PRPF19 gene in the CodAlignView genomic browser among some mammals. The positions of the first start codon of the upstream ORF in humans are indicated according to the data in Fig. 2 and stop codon (A); Alignment of protein sequences encoded by the upstream ORF in the PRPF19 gene mRNA among the indicated species in MSA4U [17]often encoding so-called leader peptides. The beginning of the protein is selected by the first possible start codon in the corresponding reading frame (B).

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6. Fig. 5. Analysis of competition between wild-type HAP1 cells and cells expressing guide RNAs over several days. The relative proportion of cells expressing guide RNA and EGFP by day three after lentiviral transduction is shown. Shown are the means and standard deviations of three independent cell infections. ** – p-value < 0.01. “vORS” – overlying ORS.

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