RNA interference induced hepatotoxicity results from loss of the first synthesized isoform of microrna-122 in mice
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1 SUPPLEMENTARY INFORMATION RNA interference induced hepatotoxicity results from loss of the first synthesized isoform of microrna-122 in mice Paul N Valdmanis, Shuo Gu, Kirk Chu, Lan Jin, Feijie Zhang, Elizabeth M Munding, Yue Zhang, Yong Huang, Huban Kutay, Kalpana Ghoshal, Leszek Lisowski, & Mark A Kay
2 Supplementary Figure 1 shrna reads specifically compete with mir-122 in Ago2- immunoprecipitated (Ago2-IP) small RNA fractions. (a) Mature and precursor raav8-u6- haat-shrnas and raav8-h1-haat-shrnas of various stem lengths in the presence (+) or absence ( ) of Dicer1 in HEK293 cells. (b) Serum haat levels for raavs expressing all combined U6-sh-hAAT-shRNAs or H1-sh-hAAT-shRNAs compared to controls (n = 9 mice per treatment except for controls where n = 3). Data are mean ± s.e.m. (c) Serum alanine transaminase (ALT) levels for each shrna construct. Values and analysis are as in (b). (d) The profile of other small RNAs in the Ago2-IP small RNA seq fraction (n = 3 mice per condition except for control where n = 1). Data are mean ± s.e.m. (e) The proportion of reads mapping to shrnas and micrornas in Ago2-IP small RNA seq fractions of mouse livers (n = 3 mice per condition except for control where n = 1). Data are mean ± s.e.m.
3 Supplementary Figure 2 Analysis of isoform biogenesis for mir-122 and other micrornas. (a) Isoforms of mir-122-5p in normal human and mouse liver samples. (b) Analysis of deposited small RNA deep sequencing data (GEO submission GSE57381) 1 of human liver samples indicates that the most predominant isoform of mir-122-5p is 22-nt in length. Mouse liver controls are from Fig. 2b (n = 4 for human liver, n = 3 for mouse). Data are mean ± s.d. (c) Quantification of reads that arise from mir-122-3p isoforms relative to total mir-122-3p reads in mouse livers receiving U6-sh-hAAT-shRNAs, H1-sh-hAAT-shRNAs or controls (n = 3 mice per condition). Data are mean ± s.e.m. (d) Rank order of the 22-nt to 21-nt ratios for micrornas that express both 22-nt and 21-nt isoforms. The position of mir-122 and other micrornas with similar isoform ratios is noted. (e) Northern blot levels of mir-107 in mouse livers do not have an obvious isoform difference of mature mir-107. (f) Northern blot analysis of mouse liver samples receiving additional U6-shRNA constructs that mimic mir-30 or target the hepatitis C virus (HCV). (g) Northern blot quantification of the mir-122-5p bands from control mouse livers and those expressing various U6-sh-RNAs. Data are mean ± s.e.m.
4 Supplementary Figure 3 The 23 rd nucleotide of mir-122-5p is added post-transcriptionally. (a) Top, mir-122-5p isoform distribution from small RNA seq after delivery of CMV-driven mir- 122 in HEK293 cells. Site directed mutagenesis of the 23 rd nucleotide to a cytosine in transfected plasmids (23U > C) still generates post-transcriptional addition of an adenine or uracil at this position. Bottom, mature mir-122-5p isoforms generated by delivered constructs that include a modified internal residue (green). (b) Small RNA seq reads corresponding to mir p after CMV-driven mir-122 delivery in HEK293 cells. Replicate numbers are the sum of samples from different time points in (a). Data are mean ± s.e.m.
5 Supplementary Figure 4 RNA seq of shrna toxic livers reveals concordant dysregulation of mrna targets. (a) Heatmap clustering of liver RNA seq samples by Fragments Per Kilobase of transcript per Million mapped reads (FPKM) values receiving either no shrnas (Liver), H1-shhAAT-shRNAs or U6-sh-hAAT-shRNAs (n = 2 mice per condition). (b) Heatmap clustering of liver RNA seq FPKM values of five additional mice receiving various U6-shRNAs and one control. (c) Ingenuity pathway analysis pathways most significantly (FDR < 0.001) altered when combining all seven U6-shRNA RNA seq samples relative to five H1-shRNA and controls. (d) RNA seq expression of major urinary protein (Mup) family member levels. (e) Combined foldchange expression of FPKM values of validated targets of mir-122-5p (n = 73) that are derepressed in mouse livers receiving U6-shRNAs. *** P < by two-tailed Student s t test.
6 Supplementary Figure 5 Loss of mir-122 leads to functional metabolic consequences including loss of Aldob expression and intolerance to a fructose rich diet. (a) Electron micrograph image reveals glycogen rich deposits (arrow) and lipid accumulation in mouse livers receiving U6-sh-hAAT-shRNAs. (b) qpcr analysis reveals that Aldoa levels increase in liver samples with reduced mir-122 levels, with a corresponding decrease in Aldob levels, commensurate with an inverse expression pattern of Aldoa and Aldob (and mir-122) in the developing liver 2-4. (c) Correlation between serum ALT levels and percent Aldob mrna expression in the liver of seven mice receiving different U6-driven shrna constructs and one control mouse. Data represent mean ± s.e.m. of 3 independent experiments in triplicate of one
7 individual mouse per condition. (d) Luciferase expression of mouse Aldoa and human ALDOA 3 UTRs and the human ALDOA 3 UTR with site directed mutagenesis of the mir-122-5p binding site. Both the 22-nt and 21-nt isoforms of mir-122-5p target this region equally. (e) Serum ALT activity showed that diets rich in fructose (60%) and glucose (60% with no fructose) corresponded to increases in ALT levels and liver toxicity (n = 5 mice per condition). (f) Mouse body weight changes following fructose rich or glucose rich diets and raav administration (n = 5 mice per condition). (g) Small RNA northern blot of mir-122-5p isoforms in mouse liver samples receiving fructose or glucose rich diets with and without U6-sh-hAAT-shRNAs. Data are mean ± s.e.m.
8 Supplementary Figure 6 Analysis of small RNA sequencing reads derived from shrnas compared to micrornas at later time points post-raav transduction. (a) The proportion of reads from U6-sh-miR-122-shRNAs and micrornas from mouse livers 8 weeks post-raav transduction. Controls are from Fig. 1f (n = 3 mice per condition). Data are mean ± s.e.m. (b) Proportion of reads from shrnas and micrornas from various raav vectors in mir-122 knockout mouse livers 3 weeks post-transduction (n = 2 mice per condition except for U6-sh- HCV where n = 1). (c) Percent of shrna reads at indicated times relative to maximum ALT levels measured over 1 month (n = 3 mice for U6-sh-miR-122 and 2 for sh-haat and sh-hcv). ULN, upper limit normal. The vertical dashed line indicates the maximum tolerated threshold of 12% in normal mouse livers. (d) Percent shrna expression in mouse liver samples 7 days after raav transduction relative to maximum serum ALT levels measured over 1 month (n = 3 mice for U6-sh-miR-122 and WT U6-sh-hAAT, n = 2 for heterozygous (het) mice and n = 1 mouse for the remaining samples). U6-sh-hAAT-25nt and U6-sh-HCV in normal liver are included from Fig. 1d for comparison purposes. Data are mean ± s.e.m. (e) Northern blot probing for mir-122 and haat shrnas in mouse livers at various time points post-raav-shrna transduction; 122ko = Mir122 knockout. (f) Proportion of reads from shrnas and micrornas in livers at indicated time points for various raav-u6-shrna constructs in mice heterozygous for Mir122.
9 Supplementary Figure 7 Model of proposed mechanism. raavs expressing U6-shRNAs compete with the first synthesized 22-nt isoform of mir-122-5p for available RNA-induced silencing complexes (RISC). This may de-repress newly synthesized mrnas that would otherwise be targeted by mir-122 leading to liver toxicity. Other micrornas are largely unaffected in this process.
10 Supplementary Figure 8 mir-122 isoforms after antisense inhibition and mrna profiles in mir-122 knockout mice (a) mir-122-5p isoform proportions in mouse liver after delivery of mir-122 or let-7 tough decoy RNAs (TuD). Data is extracted from GEO accession # GSE In each case, reads were counted for the four most abundant mir-122 isoforms and expressed as a relative percentage. mir-122 TuDs specifically diminished the 23-nt isoform of mir-122-5p leaving the 22-nt isoform relatively unchanged. (b) Mir122 knockout mice retain embryonic gene expression profiles. qrt-pcr quantification of mrna transcripts typically expressed in embryonic but not adult mouse liver (n = 14 for Mir122 knockout, n = 11 for control). *** P < by two-tailed Student s t test.
11 Supplementary Table 1 Top expressing micrornas relative to small hairpin RNA read counts. microrna U6 total average mir shrna mir mir-let-7a mir-let-7c mir-let-7f mir mir-let-7c mir-let-7f mir-let-7b mir-let-7d mmu-mir mir-let-7g mir-29a mir-26b mir mir mir U6 95% CI ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) H1 total average H1 total 95% CI ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) U6 Ago2- IP average U6 Ago2- IP 95% CI ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) H1 Ago2- IP average H1 Ago2- IP 95% CI ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) Values are presented as normalized reads per million mapped micrornas and shrnas. U6 samples include U6-sh-hAAT-21nt and U6-sh-hAAT-25nt (n = 6); H1 samples include all H1- sh-haat samples (n = 9). IP, immunoprecipitated; CI, confidence interval.
12 Supplementary Table 6 Serum lipid and glucose levels for mice receiving fructose or gluctose rich diets with or without U6-sh-hAAT-25nt shrnas. low fructose + U6- shrna high fructose + U6- shrna Chow + U6-shmiR-30 Chow + U6- shhaat- Test Referenc e range Units low fructose high fructose con Glucose mg / dl Cholesterol mg / dl Triglycerides mg / dl HDL mg / dl LDL mg / dl HDL : LDL BUN mg / dl Creatinine mg / dl Total Protein g / dl HDL, high-density lipoprotein; LDL, low-density lipoprotein; BUN, blood urea nitrogen; con, control.
13 Supplementary Table 7 List of primers used for shrna cloning, luciferase cloning and small RNA sequence alignment. sh-haat-19nt-s sh-haat-19nt-a sh-haat-21nt-s sh-haat-21nt-a sh-haat-25nt-s sh-haat-25nt-a sh-mir-122-s sh-mir-122-a sh-hcv-21-s sh-hcv-21-a sh-hcv-29-s sh-hcv-29-a sh-hcv-29b-s sh-hcv-29b-a sh-mir s sh-mir a sh-mir s sh-mir a sh- mir-30-24b-s sh-mir-30-24b-a CACCGAAGCGTTTAGGCATGTTTTCAAGAGAAACATGCCT AAACGCTTC AAAAGAAGCGTTTAGGCATGTTTCTCTTGAAAACATGCCT AAACGCTTC CACCGAAGCGTTTAGGCATGTTTAATCAAGAGTTAAACAT GCCTAAACGCTTC AAAAGAAGCGTTTAGGCATGTTTAACTCTTGATTAAACAT GCCTAAACGCTTC CACCGAAGCGTTTAGGCATGTTTAACATCTCAAGAGGATG TTAAACATGCCTAAACGCTTC AAAAGAAGCGTTTAGGCATGTTTAACATCCTCTTGAGATG TTAAACATGCCTAAACGCTTC CACCGAACGCCATTATCACACTCTATACCTGACCCATATG GAGTGTGAGAATGGTGTTTTTTGTAC AAAAACACCATTCTCACACTCCATATGGGTCAGGTATAGA GTGTGATAATGGCGTTC GATCGACTTATCCAGTTGGTTCACTCCTGACCCAAGTGAAC CAACTGGATAAGTCTTTTTGTAC AAAAGACTTATCCAGTTGGTTCACTTGGGTCAGGAGTGAA CCAACTGGATAAGTC GATCCACCGACTTATCCAGTTGGTTCACTGATGCTACCCTG ACCCAGTAGCATCAGTGAACCAACTGGATAAGTCTTTTTG TAC AAAAGACTTATCCAGTTGGTTCACTGATGCTACTGGGTCA GGGTAGCATCAGTGAACCAACTGGATAAGTC GATCCACCGACTTATCCAGTTGGTTCACTCCTGCTACCCTG ACCCAGTAGCCCAAGTGAACCAACTGGATAAGTCTTTTTG TAC AAAAGACTTATCCAGTTGGTTCACTTGGGCTACTGGGTCA GGGTAGCAGGAGTGAACCAACTGGATAAGTC GATCCACCGCTGCAAACATCCGACTGAACCCTGACCCAGT TCAGTCGGATGTTTGCAGCTTTTTGTAC AAAAGCTGCAAACATCCGACTGAACTGGGTCAGGGTTCAG TCGGATGTTTGCAGC GATCCACCGCTGCAAACATCCGACTGAAAGCTCCTGACCC AAGCTTTCAGTCGGATGTTTGCAGCTTTTTGTAC AAAAGCTGCAAACATCCGACTGAAAGCTTGGGTCAGGAGC TTTCAGTCGGATGTTTGCAGC GATCCACCGCTGCAAACATGCGACTGAAAGCTCCTGACCC AAGCTTTCAGTCGGATGTTTGCAGCTTTTTGTAC AAAAGCTGCAAACATCCGACTGAAAGCTTGGGTCAGGAGC TTTCAGTCGCATGTTTGCAGC
14 hsaldoa UTRpsiF hsaldoa UTRpsiR hsaldoa UTRmutF hsaldoa UTRmutR mmaldoa UTRpsiF mmaldoa UTRpsiR CTCGAGGCGGAGGTGTTCCCAGGCTGCCCCCAACACTCCA GGCCCTGCCCCCTCCCACTCTTGAAGA ACTAGTCTTCAAGAGTGGGAGGGGGCAGGGCCTGGAGTGT TGGGGGCAGCCTGGGAACACCTCCGCC CTCGAGGCGGAGGTGTTCCCAGGCTGCCCCCAAACCTGGA GGCCCTGCCCCCTCCCACTCTTGAAGA ACTAGTCTTCAAGAGTGGGAGGGGGCAGGGCCTCCAGGTT TGGGGGCAGCCTGGGAACACCTCCGCC CTCGAGCCAGAGCTGAACTAAGGCTGCTCCATCAACACTC CAGGCCCCTGCCTACCCACTTGCTATA ACTAGTATAGCAAGTGGGTAGGCAGGGGCCTGGAGTGTTG ATGGAGCAGCCTTAGTTCAGCTCTGGC 5 to 3 direction for each; S = sense, A = antisense. Mismatches in the mir-122 binding site of human ALDOA are underlined,
15 SUPPLEMENTARY REFERENCES 1. Selitsky, S.R., et al. Small trna-derived RNAs are increased and more abundant than micrornas in chronic hepatitis B and C. Scientific reports 5, 7675 (2015). 2. Chang, J., et al. mir-122, a mammalian liver-specific microrna, is processed from hcr mrna and may downregulate the high affinity cationic amino acid transporter CAT-1. RNA biology 1, (2004). 3. Hommes, F.A. & Draisma, M.I. The development of L- and M-type aldolases in rat liver. Biochim Biophys Acta 222, (1970). 4. Numazaki, M., Tsutsumi, K., Tsutsumi, R. & Ishikawa, K. Expression of aldolase isozyme mrnas in fetal rat liver. European journal of biochemistry / FEBS 142, (1984). 5. Xie, J., et al. Long-term, efficient inhibition of microrna function in mice using raav vectors. Nature methods 9, (2012).
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