The strong translational impact on ECM genes led us to hypothesize that the differential translation could be related to a global switch in translational control related to the generally high coding sequence (CDS) length of ECM proteins. Indeed, we observed a moderate, though highly significant correlation between CDS length and fold change (FC) in translation (r 2 = 0.26; p < 2.2 ? 10 ?16 ), which produces a downregulatory effect for genes with long CDSs and, vice versa, an upregulatory effect for genes with short CDSs (Fig. 2C). This association with CDS length was specific to heart tissue, absent in RNA-seq data, and no other genetic locus outside of the Chr. 3p teQTL showed a similar effect.
The new chromosome 3p teQTL induces polysome 50 % of-mer formation
To replicate this translatome-wide phenotype, we performed ribosome profiling on two congenic rat lines with two small, but differently sized, BN segments inserted into the short arm of Chr. 3 on an otherwise fully SHR background (see “Methods” and Fig. 2D). The first congenic line possessed a long BN segment that replaced the teQTL completely (SHR.BN-(3L)), whereas the second line contained a smaller BN segment positioned adjacent to the teQTL (SHR.BN-(3S)), hence leaving the teQTL intactparing the cardiac translatomes of both congenic lines, we fully recapitulated the protein length-dependent difference in translation observed in the HXB/BXH RI panel (r 2 = 0.20; p < 2.2 ? 10 ?16 ; Fig. 2E, F). A subsequent GO enrichment analysis on differentially translated genes concordantly yielded terms matching the downregulation of very large proteins (GO: extracellular region; padj = 6.33 ? 10 -13 ) or the upregulation of very small proteins (GO: cytosolic ribosome; padj = 1.22 ? 10 -13 ) (Fig. 2G). Of note, the observed TE fold changes specifically correlated with CDS length (r 2 = 0.20), to a lesser extent with total transcript length (r 2 = 0.162) but not with 5? UTR (r 2 = 0.004) or 3? UTR length (r 2 = 0.013) (Additional file 1: Figure S3C).
The fresh new chromosome 3p teQTL induces changes in mono- and polysome occupancy that impression stoichiometric sarcomere translation
To mechanistically dissect the brand new translational phenotype connected to the Chr. 3p teQTL, i second performed polysome profiling towards heart tissues regarding one another congenic contours (Fig. 3A). Polysome pages of SHR.BN-(3S) mice exhibited greatly altered differences in the fresh quantities of ribosomes relevant having mRNAs as compared to SHR.BN-(3L) (Fig. 3A, B and additional document step one: Contour S4A), additionally displaying quick “shoulders” associated per mono- and you will poly-ribosome height probably a sign of polysome half of-mer formation (Fig. 3C) [46, 47]. Polysome 1 / 2 of-mers is shaped in the event the 43S preinitiation complex doesn’t instantaneously join the high sixties ribosomal subunit to create a working eighties monosome. Which stand translation initiation-the speed-limiting action off RNA translation and therefore a central determinant of TE [30, forty-eight, 49]. Half-mers happen because of ribosome biogenesis problems, because of the fresh underproduction out of 1960s subunits otherwise impaired subunit signing up for [50, 51]. Yet not, design quantities of ribosomal RNA and protein elements of both ribosomal subunits checked healthy (More document 1: Contour S4B). SHR.BN-(3S) rats likewise presented increased accumulation out of highest-buy (heavy) polysomes, perhaps a sign from a problem with translation cancellation or highlighting increased translation prices regarding mRNAs with small- otherwise medium-proportions CDSs.
A great Schematic summary of the fresh new polysome fractionation and you may RNA-seq means. One to representative polysome profile per congenic rat line is offered. L, Yards, and H fractions indicate white, typical, and you can hefty polysomes, correspondingly. B Congenic range testing to possess differences in what amount of relevant ribosomes per mRNA, once the counted by the delivery regarding RNA produce along side fractions. Quantified polysome reputation urban area significantly less than curves (AUCs) can be found in More document 1: Contour S4A. Taverns imply mean opinions. C Zoomed-because of multiple polysomal highs across the replicates both for congenic lines, which have arrows exhibiting you’ll be able to half of-mers. D Heatmap with scaled RNA-seq term levels of all of the a dozen,471 quantified genetics (imply RNA FPKM ? step 1 around the replicates, for both traces).