Supplementary Figure 1: Partial DNA Sequence of the TLS Reporter Construct

Supplementary legends

Supplementary Figure 1: Partial DNA sequence of the TLS reporter construct.

A) The SV40 promoter, the multiple cloning site and the Firefly luciferase genewith a mutated start site (ATG to GCG mutation, underlined) are shown in the context of the generated luciferase reporter construct.

B) Schematic representation of the Firefly luciferase reporter construct with an inserted TLS containing a uORF and the mAUG start site plus the surrounding Kozak sequence.

Supplementary Figure 2: uORFs inhibit downstream translation of TKs in HEK cells.

A) Bar graph representing the relative luciferase activity in the presence of wt uORF and ∆uORF containing TLSs of indicated positive controls (CEBPA, CEBPB, ERBB2) and TKs, respectively.

B) Bar graph indicating the relative luciferase mRNA levels of wt uORF and ∆uORF reporter constructs for indicated TLSs of positive controls and TKs, respectively.

Error bars represent the SEM of at least twoindependent experiments. Asterisks indicate statistical significance (**p<0.01 and *p<0.05).

Supplementary Figure 3: uORF1 and the puORF regulate the translation of MET.

A) Schematic representation of the position and length of uORF1 and the puORF within the TLS of MET. SNPs that disrupt the uORF initiation codon are underlined and the resulting alternate codons are displayed in grey. Conservation of the uAUG among human and mouse (check mark) and among a total of nine vertebrate species is depicted. The weak quality of the Kozak context (-, no core Kozak base matches) is indicated.

B) Bar graph representing the relativereporter activity in the presence of the wt puORF and the ∆puORF containing TLS of MET. The translational effect of another in-frame, further upstream uORF1 was tested by mutational analysis.

C) Bar graph indicating the relative luciferase mRNA levels of the wt puORF and the ∆puORF with or without the mutation in the in-frame, more upstream uORF1 of MET.

Error bars represent the SEM of at least three independent experiments. Asterisks indicate statistical significance (**p<0.01 and *p<0.05).

Supplementary Table 1: Positions and properties of uORFs in human TK transcripts.

Gene symbols, RefSeq transcripts with TLS length and sequence, the corresponding uORFs and their properties are listed.

Supplementary Table 2: Positions and properties of uORFs in all human genes.

Gene symbols, RefSeq transcripts with TLS length, the corresponding uORFs and their properties are listed.

Supplementary Table 3: TLSs inserted into the luciferase reporter construct

Gene symbols, RefSeq transcripts and TLSs inserted into the luciferase reporter construct are listed.

Supplementary Table 4: Oligonucleotides

Primer sequences and their applications are listed.

Supplementary Table 5: SNPs in uAUGs and/or surrounding Kozak sequencecontexts of TKs.

Gene symbols, RefSeq transcripts, the affected uORFs and SNP positions are listed.

Supplementary Table 6: SNPs in uAUGs, surrounding Kozak sequence contexts and uStop codonsof all human genes.

Gene symbols, RefSeq transcripts and numbers of SNPs in uAUGs, Kozak sequence contexts and uStop codons are listed.

Supplementary Table 7: Clinical associations of SNPs in uORFs.

Gene symbols, RefSeq transcripts, SNP positions and frequencies are listed.