The circadian dynamics of small nucleolar RNA in the mouse liver

The circadian regulation of gene expression allows plants and animals to anticipate predictable environmental changes. While the influence of the circadian clock has recently been shown to extend to ribosome biogenesis, the dynamics and regulation of the many small nucleolar RNA that are required in pre-ribosomal RNA folding and modification are unknown. Using a novel computational method, we show that 18S and 28S pre-rRNA are subject to circadian regulation in a nuclear RNA sequencing time course. A population of snoRNA with circadian expression is identified that is functionally associated with rRNA modification. More generally, we find the abundance of snoRNA known to modify 18S and 28S to be inversely correlated with the abundance of their target. Cyclic patterns in the expression of a number of snoRNA indicate a coordination with rRNA maturation, potentially through an upregulation in their biogenesis, or their release from mature rRNA at the end of the previous cycle of rRNA maturation, in antiphase with the diurnal peak in pre-rRNA. Few cyclic snoRNA have cyclic host genes, indicating the action of regulatory mechanisms in addition to transcriptional activation of the host gene. For highly expressed independently transcribed snoRNA, we find a characteristic RNA polymerase II and H3K4me3 signature that correlates with mean snoRNA expression over the day.


90%
95% 100% : Similarity and identifiability of snoRNA genes. a Stacked bar chart of the numbers of snoRNA grouped into sets of equivalent genes at similarity thresholds from 85% to 100%. These sets of matched genes were built using Blast and required alignments of at least 85%, 90% and 95% of transcript length to other genes in the set (duplicate sequences were 100% similar). SnoRNA in equivalent sets are classified according to host gene biotype, designated antisense if on the opposite strand to an overlapping gene, else designated nongenic. b Observed proportion of genes in a at 85% similarity divided by the expected proportion. c Stacked bar chart of the numbers of snoRNA in a that are identifiable by a uniquely-mapping read. d Observed proportion of genes in c at 85% similarity divided by the expected proportion. snoRNA modifying(18S(rRNA Figure S3: SnoRNA modifying 18S and 28S rRNA. a Heatmap of the expression of 18S rRNA and snoRNA known to modify 18S. 18S rRNA (top row) has peak expression at ZT12-ZT16 whereas snoRNA known to interact with 18S have minimum expression at this time. Box H/ACA snoRNA are indicated by blue side colours and box C/D by green. b Fraction of snoRNA known to modify 28S whose expression is positively (solid red) and negatively (solid green) correlated with 28S expression at increasing values of R 2 (left). Similarly, for hosts of these snoRNA (right). The proportion of all genes with positive (dashed red) and negative (dashed green) correlation with 28S is shown for reference. Correlations were calculated for each gene after removing rRNA expression in each sample and rescaling to 10 6 to remove the effect of changes in rRNA on other genes across samples.  Figure S4: Cumulative fractions of genes positively and negatively correlated with pre-rRNA expression. a The cumulative fractions of snoRNA known to modify 28S rRNA whose expression was positively (red solid line) and negatively (green solid line) correlated with 28S rRNA expression at R 2 values from 0 to 1. The fractions of a reference set of genes (all other genes) with positive and negative correlations is shown for comparison (red and green dashed lines). Fewer genes have significant correlations as the R 2 threshold increases. Negative correlations with 28S are rare in the reference genes (dashed green line) whereas for snoRNA they constitute the majority of significant correlations (solid green line) The cumulative fractions of snoRNA host genes are plotted similarly b, and the cumulative fractions of snoRNA modifying 18S and their hosts are are shown in c and d. Quantification of uniquely-mapping reads log10 normalised read count at ZT16 log10 normalised read count at ZT20 a b Figure S5: Comparison of the expression of 28S-modifying snoRNA at ZT20 with expression at ZT16. a Scatterplot of snoRNA expression in TPM at ZT20 against expression at ZT16 quantified by Kallisto. b Scatterplot of snoRNA read counts at ZT20 against read counts at ZT16 quantified by uniquely-mapping reads (mapping by bowtie2, quantification by htseq-count and library depth normalised to 10 7 ). Plots shows all 28S-modifying snoRNA that could be found in the snOPY database.

Phase (h) F24
Phase (h) Nested Sampling Sf3b3 Wdyhv1 0 2 4 6 8 10 Value Figure S8: Cyclically expressed snoRNA host genes. a Heatmap of the expression of snoRNAs host genes that are inferred to be cyclic in nascent sequencing. b Heatmap of the expression of snoRNAs host genes that are inferred to be cyclic in RNA sequencing. Row side colours (white-black) represent time of peak expression (phase). c Histogram of the phase of host genes in a and in b.     Figure S11: PolII and H3K4me3 signals around clock genes. Normalised read depth in a 2kb region centered on the gene start, and oriented in the direction of transcription, is shown for clock genes Per2 and Nr1d1 for RNA Polymerase II (left) and for H3K4me3 ChIP sequencing data (right) data at seven time points.

Gm22711
Distance from gene start (base pairs)

Gm25501
Distance from gene start (base pairs)

Gm23596
Distance from gene start (base pairs)

Snora57
Distance from gene start (base pairs)

Snora17
Distance from gene start (base pairs)

Snord13
Distance from gene start (base pairs)

Gm22711
Distance from gene start (base pairs) H3K4me3 read count

Gm25501
Distance from gene start (base pairs) H3K4me3 read count

Snora57
Distance from gene start (base pairs) H3K4me3 read count

Snord13
Distance from gene start (base pairs) H3K4me3 read count Fig(S12( Figure S12: PolII and H3K4me3 signals around independently transcribed snoRNA. Normalised read depth in a 2kb region centered on the snoRNA gene start, and oriented in the direction of transcription, is shown for selected independently transcribed snoRNA for RNA Polymerase II (first and second rows) and for H3K4me3 ChIP sequencing data (third and fourth rows) data at seven time points. A peak in PolII over the gene and an adjacent peak in H3K4me3 are characteristic chromatin features. Note that Snora17 has a host gene in Refseq that is not annotated in Ensembl.  . Log2 expression over time of three snoRNA we found to be cyclic. Data is from GSE33726 and snoRNA probe mappings were taken from Ensembl GRCm38.