Variability of Human rDNA and Transcription Activity of the Ribosomal Genes
Abstract
:1. Introduction
2. Results
2.1. General Characteristics of the Observed Variability in the HT1080 Cells
2.2. Comparing Variants in the Methylated and Non-Methylated Pools
2.3. Search for Micro-RNAs (miRs) Corresponding to the Discovered Variants
- (1)
- For each studied position n we selected (from the reference database) a series of 20 bp long sequences containing n and beginning at n − 19, n − 18,…, n. These 20 sequences were subsequently entered in the database.
- (2)
- For each variable position n, we selected one 49 bp long sequence situated between n − 24 and n + 24, which was searched in the database with BLASTN and SSEARCH. Both searches were run with default parameters and a species restriction set to “human”. Search results were sorted and evaluated based on e-value; however, match length and percent identity were taken into consideration as well.
U13369.1 | CTGGGCCCGCGGCGGGCGTGGGG | (42320-42344) |
||||||||||||||||||||||| | ||
hsa-miR-6724 | CTGGGCCCGCGGCGGGCGTGGGG | (1-23) |
U13369.1 | GGAGGGAGGGAGGGAGGGAGGG | (41664-41685) |
.||.||..||||||||||...| | ||
hsa-miR-4769 | AGACGGTAGGAGGGAGGGGATG | (1-22) |
U13369.1 | TGTTCTTTCTCCCTCCC | (41831-41847) |
|||||||||||..|||| | ||
hsa-miR-6739 | GGTTCTTTCTCTTTCCC | (2-17) |
3. Discussion
- -
- by suppressing non-canonical structures generated in IGS and facilitating pRNA transcription;
- -
- by altering the structure of an miR produced in IGS;
- -
- by interfering with the activity of miRs transcribed outside the rDNA locus.
4. Materials and Methods
4.1. Cell Culture
4.2. Extraction of the Nucleoli
4.3. Methylated DNA Immunoprecipitation
4.4. Sequencing and Bioinformatic Analysis
Author Contributions
Funding
Informed Consent Statement
Conflicts of Interest
References
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Position | Variant | Frequency | Coverage | Significance | ||
---|---|---|---|---|---|---|
Methylated | Non-Methylated | Methylated | Non-Methylated | |||
41006 | insG | 0.99 | 0.99 | 41071 | 41396 | p > 0.05 |
41008 | G>C | 0.99 | 0.99 | 20577 | 20767 | p > 0.05 |
41074 | T>G | 0.16 | 0.17 | 19920 | 19203 | p > 0.05 |
41134 | A>G | 0.19 | 0.17 | 4952 | 860 | p > 0.05 |
41208 | C>T | 0.98 | 0.98 | 5317 | 920 | p > 0.05 |
41214 | T>C | 0.51 | 0.51 | 5395 | 935 | p > 0.05 |
41310 | delC | 0.95 | 0.94 | 2031 | 325 | p > 0.05 |
41479 | G>C | 0.25 | 0.23 | 2515 | 5844 | p > 0.05 |
41560 | delT | 0.99 | 0.99 | 957 | 388 | p > 0.05 |
41574 | C>T | 0.48 | 0.69 | 1195 | 4467 | p < 0.001 |
41594 | delT | 0.98 | 0.99 | 995 | 3990 | p > 0.05 |
41675 | C>A | 0.06 | 0.00 | 358 | 832 | p < 0.001 |
41679 | C>A | 0.07 | 0.00 | 289 | 489 | p < 0.001 |
41680 | C>A | 0.11 | 0.00 | 220 | 279 | p < 0.001 |
41680 | C>T | 0.41 | 0.45 | 220 | 279 | p > 0.05 |
41681 | C>A | 0.06 | 0.06 | 209 | 247 | p > 0.05 |
41781 | C>G | 0.93 | 0.88 | 166 | 290 | p > 0.05 |
41782 | G>C | 0.95 | 0.94 | 176 | 298 | p > 0.05 |
41811 | delC | 0.99 | 0.98 | 252 | 411 | p > 0.05 |
41831 | G>T | 0.05 | 0.01 | 236 | 411 | p < 0.05 |
42045 | A>C | 0.05 | 0.04 | 5578 | 21939 | p > 0.05 |
42255 | C>G | 0.99 | 0.99 | 3234 | 12700 | p > 0.05 |
42256 | G>C | 0.99 | 0.99 | 3153 | 12313 | p > 0.05 |
42301 | A>G | 0.05 | 0.06 | 1709 | 8932 | p > 0.05 |
42329 | C>A | 0.33 | 0.29 | 1221 | 9237 | p < 0.05 |
42338 | T>G | 0.05 | 0.05 | 1339 | 10181 | p > 0.05 |
42387 | A>G | 0.07 | 0.08 | 1663 | 12473 | p > 0.05 |
42400 | C>G | 0.97 | 0.95 | 1644 | 12349 | p > 0.05 |
42459 | T>C | 0.10 | 0.11 | 11659 | 12256 | p > 0.05 |
42773 | G>A | 0.05 | 0.06 | 11959 | 4163 | p > 0.05 |
42828 | T>C | 0.06 | 0.06 | 15931 | 5564 | p > 0.05 |
42945 | T>C | 0.10 | 0.10 | 13170 | 4952 | p > 0.05 |
(CCCT)n Region | Frequency | Selected Reads Coverage | Significance | ||
---|---|---|---|---|---|
Methylated | Non-Methylated | Methylated | Non-Methylated | ||
Q1 (41667–41686) | 0.95 | 0.11 | 62 | 18 | p < 0.0001 |
Q2 (41842–41877) | 0.98 | 1.00 | 83 | 179 | p > 0.05 |
Region | Length | Frequency |
---|---|---|
Q1 | 12 | 0.47 |
32 | 0.18 | |
28 | 0.20 | |
24 | 0.04 | |
16 | 0.07 | |
20 | 0.02 | |
1 | 0.02 | |
24 | 0.51 | |
Q2 | 8 | 0.26 |
28 | 0.18 | |
12 | 0.03 | |
7 | 0.01 | |
4 | 0.01 | |
1 | 0.01 | |
Q1 + Q2 | 24 | 0.42 |
8 | 0.22 | |
28 | 0.18 | |
12 | 0.11 | |
32 | 0.03 | |
7 | 0.01 | |
4 | 0.01 | |
1 | 0.01 | |
20 | 0.00 |
miR Name | Orientation | Origin | SNV |
---|---|---|---|
hsa-miR-6724 | Sense | IGS | 42329, C>A |
hsa-miR-4769 | anti-sense | outside rDNA | 41675, C>A 41679, C>A 41680, C>A |
hsa-miR-6739 | anti-sense | outside rDNA | 41831, G>T |
Position | Size of the Product (bp) | Forward Primer | Reverse Primer |
---|---|---|---|
40877–41134 | 257 | GGCTTGGCTGATGTTTGTG | GCAGAGATACACGTTGTCGT |
41051–41358 | 307 | TCGTTTCCACGCGTTTAC | CACGAAGGAGAGGAAGAAAG |
41311–41568 | 275 | CCGAGCGTACGTAGTTATCTC | AAGACAGACGGGAAGGAAAG |
41469–41790 | 321 | CGTTCCCTGTGTTTCCTTCT | GCAGAATCGGTAGGCTCTTC |
41748–42033 | 285 | GTCTGTCTCTGCGTGGATTC | CGAAACCGTGAGTCGAGAAG |
41953–42323 | 370 | TTTGGGCACCGTTTGTGT | CAGAAGCGCAGCGACAG |
42276–42607 | 332 | TCTGGCGTGCAGGTTTATGT | GGACTCGCCAGAAAGGATCG |
42589–43029 | 439 | GATCCTTTCTGGCGAGTCC | ACAGGTCGCCAGAGGACAG |
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Chmúrčiaková, N.; Smirnov, E.; Kurfürst, J.; Liška, F.; Cmarko, D. Variability of Human rDNA and Transcription Activity of the Ribosomal Genes. Int. J. Mol. Sci. 2022, 23, 15195. https://doi.org/10.3390/ijms232315195
Chmúrčiaková N, Smirnov E, Kurfürst J, Liška F, Cmarko D. Variability of Human rDNA and Transcription Activity of the Ribosomal Genes. International Journal of Molecular Sciences. 2022; 23(23):15195. https://doi.org/10.3390/ijms232315195
Chicago/Turabian StyleChmúrčiaková, Nikola, Evgeny Smirnov, Jaroslav Kurfürst, František Liška, and Dušan Cmarko. 2022. "Variability of Human rDNA and Transcription Activity of the Ribosomal Genes" International Journal of Molecular Sciences 23, no. 23: 15195. https://doi.org/10.3390/ijms232315195