Supplemental Figure S1. Expression of Cirbp mrna in mouse tissues and NIH3T3 cells.

Size: px

Start display at page:

Download "Supplemental Figure S1. Expression of Cirbp mrna in mouse tissues and NIH3T3 cells."

Victor Goodwin
5 years ago
Views:

1 SUPPLEMENTAL FIGURE AND TABLE LEGENDS Supplemental Figure S1. Expression of Cirbp mrna in mouse tissues and NIH3T3 cells. A) Cirbp mrna expression levels in various mouse tissues collected around the clock during 2 consecutive days. Light and gray areas reflect subjective days and nights, respectively. B) Polar plot showing phases and daily fold changes (log2 FC) of Cirbp expression in different tissues. The red-blue gradient circle represents mouse CBT values around the clock, with red and blue symbolizing high and low temperatures, respectively. The data used for generating the diagrams shown in panels A) and B) were extracted from (Zhang et al., 2014). C) Temporal Cirbp mrna and pre-mrna accumulations (represented in RPKM) in liver samples collected around the clock from mice fed ad libitum (Atger et al., 2015). D) Autoradiograph of the Cirbp 5 UTR RPA with pooled T7 polymerase-amplified crna samples collected in 3 experiments from cells exposed to simulated amplified body temperature cycles (33.5⁰C ⁰C). The Cirbp RNA probe was designed to protect the bp (exon 1 - exon 3) of the major mrna transcript (and the remaining parts of the putative truncated isoforms) and the additional upstream sequence of 63 bp of the longer putative alternative isoform. ZT: Zeitgeber Time. Semiquantitative PCR analysis of Cirbp 5 RACE samples from NIH3T3 cells incubated at 37⁰C and 32⁰C for various time periods (E) and of Cirbp 3 RACE samples collected 6 h after the temperature shift (F). major Cirbp isoform; * Cirbp precursor with un-spliced intron 6. The seemingly higher expression of this transcript at 38⁰C was found to be insignificant by other more quantitative methods discussed in the main text. The apparently temperature-dependent levels of the shorter DNA fragments (within the brackets) were not reproducible and likely represent PCR artifacts. Images of 3 independent experiments are shown in each panel.

2 Supplemental Figure S2. CMV-LMLucR construct expression analysis. A) qpcr analysis of LMLucR mrna levels at minimum (35.5⁰C) and maximum (38.5⁰C) temperature values (lower panel, red arrows) in a NIH3T3 stable cell line expressing a CMV-LMLucR transgene. Data are normalized to Ppib levels and presented as FC between the sample and the 38.5⁰C sample. (1 to 3: biological replicates). B) Example of how bioluminescence background was removed from raw data. Upper panel: The raw bioluminescence count track (black) is fitted with its baseline curve (green). Lower panel: The red curve was obtained by calculating the ratios between measured photon counts (black in upper panel) and estimated background values (green in upper panel). Supplemental Figure S3. mrna stability cannot account for temperature-dependent Cirbp mrna accumulation. Representative images of RNase protection assays with T7- amplified crna samples collected at different time points after temperature down-shift (38⁰C 33⁰C) (A) and up-shift (33⁰C 38⁰C) (B). The probes used were the same Cirbp intron 6 - exon 7 and Ppib exon 6 probes as in Figure 1. C) Quantification of the RPA experiment. Black circles represent the measured Cirbp signals in two biological replicates, black lines the calculated fits and red dotted lines indicate the half-lives at the two temperatures. The shaded gray areas indicate two standard deviations (calculated from the raw data) around the fit. D) The simplest model describing pre-mrna and mrna dynamics. The transcription rate is denoted as k s, pre-mrnas decay with a rate k p and processing (splicing) of pre-mrnas into mature mrnas occurs at the rate ρ. Finally, the mrnas are degraded at the rate k m. E) A more complex model than that shown in (D). In model (E) pre-mrnas exist in two states: pre-mrnas prone to degradation and pre-mrnas prone to splicing. In the full form of the model (Supplemental Materials and Methods), shown on the upper part of the cartoon, a premrna molecule can reversibly switch between the two states. Assuming fast switching, the model reduces to the lower part of the cartoon, where a single parameter α controls the

3 fraction of splice-prone pre-mrnas in the total pool of pre-mrnas (Supplemental Materials and Methods). The rest of the reactions in this model are identical to those of model (D). F) Graphs show the measured qpcr values for the Cirbp intron 1 amplicon (green symbols). The green lines show the prediction from the mathematical model (independently fitted to the RNA-seq experiment in Figure 4). The shaded green areas represent the estimated uncertainty of the fit. G) qpcr analysis of the LMlucR amplicon levels in 3T3 Flp-In stable cell lines expressing gcirbp-lmlucr and Cirbp-cDNA-LMLucR transgenes. Cells were seeded and kept for 24 h at 37⁰C to avoid the potential confounding response of the CMV promoter to the serum change. The cells were then incubated at 33⁰C or 38⁰C for 13 h and shifted to the opposite temperature for 9 h. n 3 biological replicates for each. Data are normalized to endogenous Ppib levels and presented as FCs between a particular sample at 38⁰C and 33⁰C. Bars represent SDs. (*) p<0.05, t-test assuming unequal variances. Supplemental Figure S4. Temperature-dependent Cirbp degradation pathways do not involve the usual degradation machinery. A) qpcr analysis (left panels) of Cirbp and Lgals1 transcripts in AMO-treated (2.5 nmol of Cirbp intron 4 - exon 5 and Lgals1 intron 1 - exon 2 AMO oligonucleotide each) and control (5 nmol of Standard control oligo) samples 16 h after temperature shift/transfection. n=2 series of 3 biological replicates; data were normalized to endogenous Ppib mrna levels and presented as FCs between a particular sample and a control 38⁰C sample of the same series; (*) p<0.05, t-test assuming unequal variances. (Right panel) Gel image of semiq. PCR analysis of Cirbp transcripts in AMOtreated and control samples. * constitutive Cirbp mrna ( ); shorter splice isoform accumulating in AMO-treated cells. qpcr analysis of Cirbp mrna and pre-mrna levels in cells transfected with sirnas directed against Xrn2 (B), Dom3z (C), Exosc9 (D), Exosc10 (E) and Xrn1 (F) transcripts. n 3 biological replicates for each. Data are normalized to

4 endogenous Ppib levels and presented as FCs between a particular sample and the average value of 38⁰C control samples. Bars represent SDs. Supplemental Figure S5. Splicing-dependent regulatory element in Cirbp intron 1 enables temperature-mediated Cirbp expression. (A) Schematic representation of Cirbpluciferase reporter constructs with (+) or without (-) temperature-dependent expression. (B) Bioluminescence tracks of the gcirp-lmlucr (construct A) and LMLucR-gCirbp (E) reporter constructs in transiently transfected cells. Representative image of n 3 experiments. C) Representative semi-qpcr gel image of LMLucR-Cirbp amplicons in samples collected from cells transiently transfected with LMLucR-gCirbp construct (E) incubated at 33⁰C and 38⁰C. Arrow - full length LMLucR F + Cirbp 5 UTR R amplicon; arrowhead & asterisk - amplicons stemming from transcripts arisen through alternative use of splice donor sites in the LMLucR gene. Supplemental Figure S6. Genome-wide analysis by RNA-seq reveals a role of temperature in regulating pre-mrna splicing efficiency and mrna stability. A) Identification of thermo-sensitive genes. Number of identified thermo-sensitive genes as a function of the cutoff value for the minimum mrna (log2 FC relative to time zero), and a p- value cutoff (marked on the different columns, calculated by edger). In this study we used 0.5 as the minimum allowed mrna log2 FC cutoff and p<0.05, which resulted in the identification of 201 thermo-sensitive genes. B) Examples of mrna and pre-mrna expression profiles at the two temperatures for cold-inducible (Nxpe4) and heat-inducible (Rnf151) genes, both of which reached a new steady-state. The black circles and green triangles denote the mrna and pre-mrna measurements, respectively. The solid lines show the best fit and the shaded area its uncertainty range. C) Among the thermo-sensitive genes we identified a subset of transiently responding genes (Supplemental Table S5, details in

5 Supplemental Materials and Methods). Here we show the temporal mrna and pre-mrna accumulation profiles for two transiently induced genes, with Txnrd3 and Hsp90 representing a cold- and heat-inducible gene, respectively. The description of the symbols is the same as that used in B). Supplemental Figure S7. RNA-seq data analysis. A) Normalization of RNA-seq data at 38⁰C and 33⁰C to the total number of exon-exon junction reads in each library for the two biological replicates (rep1 and rep2). Fold changes (FC) of pre-mrna (green boxes) and mrna (violet boxes) transcripts at each time point (t1-9h) were normalized to the time point 0. As expected, for the large majority of genes temperature did not affect mrna and premrna accumulation. Hence, the mean FC for mrna and pre-mrna is centered near zero, marked by the horizontal dashed line. B) DNase I hypersensitivity site profiles (black) from the modencode project (Celniker et al., 2009) for six mouse tissues at the Cirbp locus shown with its RNA expression track in blue. The reads from the DNase I hypersensitive site (DHS) within the first intron of Cirbp (red-shaded area) were omitted for the quantification of pre-mrna transcripts (see Supplemental Materials and Methods). Supplemental Table Legends for Excel Files Supplemental Table S4. Splice site and functional RNA motif analysis of the Cirbp premrna sequence by the ASSP and RegRNA.2 algorithms. Supplemental Table S5. Lists of temperature-dependent transcripts and RNA processing steps identified by the ATSS RNA-seq analysis. Tabs 1-6: Relative abundances of mrnas and pre-mrnas for cold-inducible (C-IN), heat inducible (H-IN) and transiently temperature-responsive transcripts. Tabs 7 and 8: Probabilities that the indicated model

6 parameters are changed following the temperature shifts for cold- and heat-inducible CIRBPbinding and non-binding transcripts. Supplemental Table S6. Gene ontology analysis for the set of cold-inducible and heatinducible genes performed by the DAVID Bioinformatics Resources algorithm. Supplemental Table S8. List of oligonucleotide sequences. Supplemental Table S9. List of reporter constructs and cloning strategies.

Nature Genetics: doi: /ng.3731

Nature Genetics: doi: /ng.3731 Supplementary Figure 1 Circadian profiles of Adarb1 transcript and ADARB1 protein in mouse tissues. (a) Overlap of rhythmic transcripts identified in the previous transcriptome analyses. The mouse liver