(A) Regulation mode of SRGs by B2 RNA processing based on previous works (Zovoilis et al., 2016; Yakovchuk et al., 2009; Ponicsan et al., 2010). Color intensity represents higher B2 RNA binding …
(A) Experimental design for study of B2-SRGs in the hippocampus of the amyloid pathology mouse model (APP) and the respective wild type (WT) control. (B) Immunohistochemistry for identifying …
(A–B) Expression levels comparison between RNA-seq data from WT 6-month-old mouse hippocampus used in our study (panel A) and RNA-seq data from the Allen Brain Atlas (Panel B). Comparison is done …
(A) Expression levels calculated through RNA-seq for the selected genes to be tested through RT-qPCR in (B). These are 12 from the genes that were found to be upregulated in APP 6-month-old mice …
(A) Experimental design for study of B2-SRGs in the hippocampus of the amyloid pathology mouse model (APP) and the respective wild type (WT) control. (B) Expression levels of non-B2 RNA regulated …
(A) Plotting of the position of the first base (5′ end) of B2 RNA fragments across the B2 loci to depict increased levels of B2 RNA fragments in 6-month-old APP mice. Upper panel: Secondary …
This figure relates to Figure 3A. Upper panel: As in Figure 3A. Secondary structure and processing points of B2 RNA. Secondary structure of B2 RNA adapted from Espinosa and colleagues (Espinoza et …
(A) Boxplot depicts distribution of expression levels of Hsf1 gene among different age groups of mice between wild type and APP. Values are based on TPM counts of long-RNA-seq data. Statistical …
(A) Mouse cortex and hippocampus gene expression levels for Ezh2 and Hsf1 depicted in the Allen Brain Atlas Transcriptomics explorer showing limited expression of Ezh2 across multiple neural tissues …
(A) In vitro incubation of B2 RNA. In vitro transcribed and folded B2 RNA at 200 nM incubated with PNK as a control (lane 1), 250 nM Hsf1 (lane 2) and without protein (lane 4). Incubations occurred …
Full gel images for Figure 5, part 1.
Full gel images for Figure 5, part 2.
(A) Plotting of the position of the first base (5′ end) of B2 RNA fragments across the B2 loci produced by B2 RNA that has been processed in vitro in the presence of Hsf1. Mapping was done as in …
(A) Experimental design for the amyloid toxicity cell culture assay employing HT-22 cells. Cells culture media were supplemented with Fetal Calf Serum (FCS) and the scramble LNA described in Figure 8…
Plots are based on amyloid pathology genes (Supplementary file 1). Correlation matrix was constructed using Semonk for the reads per million per gene length counts.
(A) Experimental design for the B2 RNA knock-down cell culture assay employing HT-22 cells. (B) Expression levels of full-length B2 RNA (RT-qPCR) in the B2 RNA KD experiment. Statistical …
(A) Experimental design of the combined Hsf1 Knock Down – amyloid toxicity assay in HT22 cells followed by short and long RNA-seq. (B) Expression levels of Hsf1 as defined by long-RNA-seq (upper …
(A) Experimental design for estimation of B2 RNA processing ratio based on short and long-RNA seq data in our HT22 amyloid toxicity model. (B) Boxplot depicts distribution of levels of processed …
(A) Experimental design of the combined Hsf1 Knock Down – amyloid toxicity assay in HT22 cells followed by short- and long-RNA-seq. (B) Expression levels of non-B2 RNA regulated genes as defined by …
Upon removal of the stress-generating stimulus, healthy cells restore the expression levels of Hsf1, specific B2 RNA regulated target genes and processing ratio of B2 RNAs returns to base levels. In …
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Cell line (Mus musculus) | HT-22 | Millipore Sigma | Cat#SCC129, RRID:CVCL_0321 | |
Sequence-based reagent | Amyloid Beta peptides ( 1-42) | Sigma-Aldrich | Custom synthesis | DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA |
Sequence-based reagent | Amyloid Beta peptides (Reverse 42–1) | Sigma-Aldrich | Custom synthesis | AIVVGGVMLGIIAGKNSGVDEAFFVLKQHHVEYGSDHRFEAD |
Peptide, recombinant protein | Hsf1 protein | Enzo life sciences | ADI-SPP-902-F | Synthesized in insect, human sequence |
Commercial assay, kit | NEBNext Small RNA Library Prep set | NEB | Cat# E7330 | |
Commercial assay, kit | NEBNext Ultra II directional RNA library prep kit | NEB | Cat# E7760 | |
Commercial assay, kit | Superscript III RT | Invitrogen | Cat# 18080093 | |
Commercial assay, kit | Luna universal master mix | NEB | Cat# M3003 | |
Antibody | Anti-Hsf1 (Rabbit, polyclonal) | Enzo | Cat# ADI-SPA-901 RRID:AB_10616511 | WB: 1:1000 |
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Sequence-based reagent | FosB Forward | IDT | Custom | 5-CGAGCTGCAAAAAGAGAAGG −3 |
Sequence-based reagent | FosB Reverse | IDT | Custom | 5- TTACAGAGCAAGAAGGGAGG −3 |
Sequence-based reagent | Pag1 Forward | IDT | Custom | 5-GAGCACAACTTCAAAGCTGG-3 |
Sequence-based reagent | Pag1 Reverse | IDT | Custom | 5- TCATCAGGTTCTCATGGTCC −3 |
Sequence-based reagent | Sema5a Forward | IDT | Custom | 5- ATGAGGCTGTGCAGTTCAGT-3 |
Sequence-based reagent | Sema5a Reverse | IDT | Custom | 5-GTAACCAGGGGCCAATTTCT-3 |
Sequence-based reagent | Sgms1 Forward | IDT | Custom | 5- ACCATAGACCACACAGGCTA-3 |
Sequence-based reagent | Sgms1 Reverse | IDT | Custom | 5- TTTCTTCCGGTCTGAGCACT-3 |
Sequence-based reagent | Hsf1 Forward | IDT | Custom | 5- TGACACCGAGTTCCAGCATC-3 |
Sequence-based reagent | Hsf1 Reverse | IDT | Custom | 5- TGACACTGTCCTGGCGTATT-3 |
Sequence-based reagent | Mitf Forward | IDT | Custom | 5- AAGCTCAGAGGCACCAGGTA-3 |
Sequence-based reagent | Mitf Reverse | IDT | Custom | 5- CCTGCTCTGCTCCTCAAACT-3 |
Sequence-based reagent | 7SK Forward | IDT | Custom | 5-GACATCTGTCACCCCATTGA-3 |
Sequence-based reagent | 7SK Reverse | IDT | Custom | 5- GCCTCATTTGGATGTGTCTG-3 |
Sequence-based reagent | Hprt Forward | IDT | Custom | 5- TCCTCCTCAGACCGCTTTT-3 |
Sequence-based reagent | Hprt Reverse | IDT | Custom | 5- CCTGGTTCATCATCGCTAATC-3 |
Sequence-based reagent | B2 Forward | IDT | Custom | 5- GGGGCTGGTGAGATG-3 |
Sequence-based reagent | B2 Reverse | IDT | Custom | 5-AGCTGTCTTCAGACACTCC −3 |
Sequence-based reagent | Adcy1 Forward | IDT | Custom | 5- GCATGACAATGTGAGCATCC −3 |
Sequence-based reagent | Adcy1 Reverse | IDT | Custom | 5-TCAAGTCCCATCTCCACACA −3 |
Sequence-based reagent | Kcnq3 Forward | IDT | Custom | 5- AGCACCGTCAGAAGCACTTT −3 |
Sequence-based reagent | Kcnq3 Reverse | IDT | Custom | 5-TCCAAGAGACCCAGCTTTTG-3 |
Sequence-based reagent | Klf15 Forward | IDT | Custom | 5-TCATGGAGGAGAGCCTCTGT-3 |
Sequence-based reagent | Klf15 Reverse | IDT | Custom | 5-TCCAAGAGACCCAGCTTTTG-3 |
Sequence-based reagent | Magi2 Forward | IDT | Custom | 5-CGGGATCACACTTTTCACCT-3 |
Sequence-based reagent | Magi2 Reverse | IDT | Custom | 5-CGGGATCACACTTTTCACCT-3 |
Sequence-based reagent | Palld Forward | IDT | Custom | 5-CAGTGGCTCAGACAGCACAT-3 |
Sequence-based reagent | Palld Reverse | IDT | Custom | 5-CTCCTGTTTTCGGAGCTGAG-3 |
Sequence-based reagent | Enpp2 Forward | IDT | Custom | 5-GACTGTCGGTGTGACAACCT-3 |
Sequence-based reagent | Enpp2 Reverse | IDT | Custom | 5-CTTCTGAGCAGTGACAGGCA-3 |
Sequence-based reagent | RPS15 Forward | IDT | Custom | 5-AACCAGAGATGATCGGCCAC-3 |
Sequence-based reagent | RPS15 Reverse | IDT | Custom | 5-ATGAATCGGGAGGAGTGGGT-3 |
Sequence-based reagent | Calm2 Forward | IDT | Custom | 5-GACTGAAGAGCAGATTGCAG-3 |
Sequence-based reagent | Calm2 Reverse | IDT | Custom | 5-CAGTTCTGCTTCTGTGGGGT-3 |
Sequence-based reagent | Kalrn Forward | IDT | Custom | 5-CCCTGAACTCCATCCACAGT-3 |
Sequence-based reagent | Kalrn Reverse | IDT | Custom | 5-GAGGGGTGTGTGTGACTCTT-3 |
Sequence-based reagent | B2 RNA | IDT G-block | Custom synthesis. Zovoilis et al., 2016 | 5′- taatacgactcactata GGGGCTGGTGAGATGGCTCAGTGGGTAAGAGCACCCGACTGCTCTTCCGAAGGTCCGGAGTTCAAATCCCAGCAACCACATGGTGGCTCACAACCATCCGTAACGAGATCTGACTCCCTCTTCTGGAGTGTCTGAAGACAGCTACAGTGTACTTACATATAATAAATAAATAAATCTTTAAAAAAAAA - 3 |
Sequence-based reagent | B2mut4b | IDT G-block | Custom synthesis. | taatacgactcactataGGGCTGGTGAGATGGCTCAGTGGGTAAGAGCACCCGACTGCTCTTCCGAAGGTCCGGAGTTCAAATCCCAGCAACCACATGGTGGCTCACAACCATCCGTAACGAGATCTGACTCCCTCTTCTTCTGAAGACAGCTACAGTGTACTTACATATAATAAATAAATAAATCTTTAAAAAAAAA |
Supplementary tables.
Supplementary Table 1. List of B2 RNA regulated SRGs(B2-SRGs). Data are compiled from Zovoilis et al., 2016 and include those genes that are close to B2 CHART peaks (genome-binding sites) before but not after the application of stress stimulus. Supplementary Table 2. Complete lists of enriched terms in B2 RNA regulated SRGs(B2-SRGs)(see Suppl.Table 1) for Tissue Enrichemnt (left), Biological Process (middle) and Cellular Compartment (right). Supplementary Table 3. List of B2 RNA regulated SRGs (B2-SRGs) (see Suppl.Table 1) that are associated with learning based on Peleg et al., 2010. Supplementary Table 4. Upregulated genes in hippocampi of APP 6-month-old mice compared to 6-month WT mice. Values were calculated using DESeq (see Materials and methods) on long-RNA-seq data. Only genes with an FDR < 0.2 are depicted. Supplementary Table 5. List of B2 RNA regulated SRGs (B2-SRGs) (see Suppl.Table 1) that are upregulated in 6-month-old APP mice compared to WT (see Suppl.Table 4) Supplementary Table 6. List of B2 RNA regulated SRGs (B2-SRGs) (see Suppl.Table 1) that are upregulated in 6-month-old APP mice (see Suppl.Table 4) and are associated with learning based on Peleg et al., 2010. Supplementary Table 7. Complete lists of enriched terms in B2 RNA regulated SRGs (B2-SRGs) that are upregulated in 6-month-old APP mice compared to WT (see Suppl.Table 5) for Biological Process (left) and Cellular Compartment (right). Supplementary Table 8. Upregulated genes in HT22 cells treated with amyloid beta and Scr LNA compared to cells treated with the control peptide and scr LNA. Values were calculated using DESeq (see Materials and methods) on long-RNA-seq data. Only genes with an FDR < 0.2 are depicted. Supplementary Table 9. List of genes that are upregulated in HT22 cells treated with amyloid beta (see Suppl.Table 8) and in 6-month-old APP mice (see Suppl.Table 4) Supplementary Table 10. List of B2 RNA regulated SRGs (B2-SRGs) (see Suppl.Table 1) that are upregulated in HT22 cells treated with amyloid beta and Scr LNA compared with cells treated with the control peptide and scr LNA (see Suppl.Table 8 ) Supplementary Table 11. Complete lists of enriched terms in B2 RNA regulated SRGs (B2-SRGs) that are upregulated in HT22 cells treated with amyloid beta (see Suppl.Table 10) for Biological Process (left) and Cellular Compartment (right). Supplementary Table 12. Correlation co-efficients and p-values for genes of Figure 8—figure supplement 2. Includes genes for which there was readcoverage across all sample and the correlation p value was less than 0.05. Supplementary Table 13. List of non-B2 RNA regulated genes (random set) used throughout the study.