Suppression of RNA Recognition by Toll-like Receptors: The Impact of Nucleoside Modification and the Evolutionary Origin of RNA
Immunity, 2005 · paper · Biomedicine
Studies how modified nucleosides in RNA affect immune recognition by Toll-like receptors. It finds that modifications such as pseudouridine suppress activation of dendritic cells, while bacterial RNA, lacking modifications, activates them strongly.
5 findings
Substance Results state "When RNAs with modified nucleoside levels of 1.7%–3.2%, which correspond to 14–29 modifications per molecule, were tested, the RNA could maintain only half of its capacity to induce expression of TNF-α", pairing the expected-percentage row of Figure 5B with the measured-number row; 14–29 residues of a 1571-nt RNA is about 0.9%–1.8%.
- p. 5 Results, "Suppression of RNA-Mediated Immune Stimulation…" (left column, bottom)
modified nucleoside levels of 1.7%– 3.2%, which correspond to 14–29 modifications per molecule
- p. 5 Results, preceding sentence
0.2%–0.4% m6A, Ψ, or m5C in the RNA, which corresponds to approximately three to six modified nucleosides per one molecule of the 1571 nt-long RNA
- p. 7 Figure 5 legend (B)
the nucleoside content of RNA-1571 (A: 505, U: 451, C: 273, and G: 342)
- p. 5 Results, "Suppression of RNA-Mediated Immune Stimulation..."
When RNAs with modified nucleoside levels of 1.7%–3.2%, which correspond to 14–29 modifications per molecule, were tested
Only the authors can say what is right here.
Judge's note: Read the Figure 5B table on the p007 page image and crops C011-c and C015-b, and the Results sentences on p005 crops C011-e and C012-b. Figure 5B at the 10% reaction ratio reads: expected (%) m6A 3.2, Ψ 2.9, m5C 1.7; measured (%) m6A 0.9, Ψ 1.9, m5C 1.4; measured (number) m6A 14, Ψ 29, m5C 23. So "1.7%–3.2%" is the expected-(%) range and "14–29" is the measured-number range for the same samples, whose measured percentages are 0.9–1.9. The legend confirms the composition A:505, U:451, C:273, G:342, summing to 1571. Recomputing: 14 of 1571 is 0.89% and 29 of 1571 is 1.85%; conversely 1.7% and 3.2% of 1571 are about 27 and 50 residues. I checked the alternative denominator of the corresponding unmodified nucleoside (14/505, 29/451, 23/273) and it does not give 1.7–3.2% either. The immediately preceding sentence uses the measured row consistently (0.2%–0.4% with three to six residues; 0.002 x 1571 = 3.1, 0.004 x 1571 = 6.3), so the paper switches convention without saying so and the stated correspondence does not hold as printed. I found no footnote or methods statement reconciling the two rows. Only the authors can say whether the percentages or the counts should change, hence author_query.
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Reference or label Figure 1 legend enumerates the treatments as "lipofectin alone, or complexed with R-848 (1 µg/ml), or RNA (5 µg/ml) from 293 cells (total, nuclear, and cytoplasmic RNAs), mouse heart (polyA+ mRNA), human platelet mitochondrial RNA, bovine tRNA, bacterial tRNA, and total RNA (E. coli)" but omits two plotted conditions, "poly(I):(C)" and "in vitro transcribed RNA-1866".
- p. 2 Figure 1 legend
Human MDDCs were incubated with lipofectin alone, or complexed with R-848 (1 g/ml), or RNA (5 g/ml) from 293 cells (total, nuclear, and cytoplasmic RNAs), mouse heart (polyA+ mRNA), human platelet mitochondrial RNA, bovine tRNA, bacterial tRNA, and total RNA (E. coli)
- p. 2 Figure 1, x-axis category labels
in vitro transcribed RNA-1866
Judge's note: Read the p002 page image. The Figure 1 x-axis carries, left to right: lipofectin, poly(I):(C), R-848, in vitro transcribed RNA-1866, then the bracketed "mammalian RNA" group (total, nuclear, cytoplasmic, polyA+ mRNA, mitochondrial, tRNA) and the bracketed "E. coli RNA" group (total, tRNA). The legend as printed lists lipofectin, R-848 and the RNA sources but never mentions poly(I):(C) or the in vitro transcript. The in vitro transcript is the comparator the Results rely on ("at very low levels relative to RNA synthesized in vitro by T7 RNA polymerase (RNAP) (Figure 1)"), and poly(I):(C) is a separately purchased reagent per the Methods on p010, so it is not covered by the generic "RNA (5 µg/ml)" clause. The legend is incomplete with respect to the data plotted.
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Reference or label Figure 4B heads its treatment series "RNA-1866" while Figure 4A heads the identical series "RNA-1571" and the legend states "Modified nucleosides present in RNA-1571 are indicated."
- p. 3 Figure 4B, table row heading
RNA-1866
- p. 6 Figure 4A, header bar above the six lower dot plots
RNA-1571
- p. 6 Figure 4 legend
Modified nucleosides present in RNA-1571 are indicated.
- p. 4 Results, "Modified Nucleosides Reduce the Capacity of RNA…"
FACS analysis of MDDCs treated with RNA-1571 and its modified versions
Only the authors can say what is right here.
Judge's note: Read the full p006 page image. Panel A has a bar labelled "RNA-1571" spanning the six lower dot plots headed unmodified, m5C, m6A, Ψ, s2U, m6A/Ψ. Panel B is a table with rows lipofectin, poly(I):(C), R848, then an italic group headed "RNA-1866" containing exactly the same six entries (unmodified, m5C, m6A, Ψ, s2U, m6A/Ψ) with TNF-α pg/ml and CD80/CD86 mean fluorescence. The legend (crop C013-b and p006) says only "Modified nucleosides present in RNA-1571 are indicated", and the Results on p004 attribute the whole of Figure 4 to RNA-1571 ("FACS analysis of MDDCs treated with RNA-1571 and its modified versions revealed that modified nucleosides such as m5C, m6A, Ψ, s2U, and m6A/Ψ decrease the ability of RNAs to induce cell surface expression of CD80, CD83, CD86, and MHC class II (Figure 4)"). Methods on p010 define RNA-1866 and RNA-1571 as distinct transcripts, so this is not an alternative name. Both labels are legible in the image, so this is not an extraction artefact. Only the authors can say which label is the correct one, hence author_query.
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Reference or label Results cite Figure 5B twice for TNF-α inhibition data — "proportionally inhibited the capacity of RNA to induce TNF-α (Figure 5B)" and "detectable inhibition of cytokine secretion (Figure 5B)" — but those measurements are plotted in Figure 5C; Figure 5B is the nucleoside-content table.
- p. 5 Results, "Suppression of RNA-Mediated Immune Stimulation..."
proportionally inhibited the capacity of RNA to induce TNF-α (Figure 5B)
- p. 5 Results, same paragraph
detectable inhibition of cytokine secretion (Figure 5B)
- p. 7 Figure 5 legend
(C) MDDCs were transfected with lipofectin-complexed capped RNA-1571 (5 g/ml) containing the indicated amount of m6A, Ψ, or m5C. After 8 hr, TNF-α was measured in the supernatants by ELISA.
- p. 4 Results, proportional suppression
proportionally inhibited the capacity of RNA to induce TNF-α (Figure 5B)
Judge's note: Read p005 crops C011-e and C012-b for the two citations, and the p007 page image plus crops C011-c and C015-b for Figure 5. Panel B is a table of "Modified nucleoside in reaction (%)" with expected (%), measured (%) and measured (number) rows, containing no cytokine data. Panel C is the bar chart with x-axis "Relative inhibition of TNF-α expression (%)" and y-axis "Number of modified nucleosides in RNA-1571", and its legend (crop C012-c) reads "(C) MDDCs were transfected with lipofectin-complexed capped RNA-1571 (5 µg/ml) containing the indicated amount of m6A, Ψ, or m5C. After 8 hr, TNF-α was measured in the supernatants by ELISA." The Discussion on p008 cites the same result correctly as "(Figure 5C)". Both Results pointers should be to panel C.
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Wording Summary states "We show that RNA signals through human TLR3, TLR7, and TLR8, but incorporation of modified nucleosides m5C, m6A, m5U, s2U, or pseudouridine ablates activity", but the Results and Discussion restrict TLR3 ablation to m6A and s2U and state that Ψ-modified RNA "could potently activate TLR3".
- p. 1 Summary
We show that RNA signals through human TLR3, TLR7, and TLR8, but incorporation of modified nucleosides m5C, m6A, m5U, s2U, or pseudouridine ablates activity.
- p. 3 Results, right column
The presence of other nucleoside modifications such as m5C, m5U, Ψ, or m5C/Ψ in the RNA had a less remarkable suppression or no effect at all on the potential of RNA to activate TLR3
- p. 4 Results, left column
Specifically, m6A and s2U modifications suppress the ability of RNA to stimulate TLR3, whereas m6A, m5C, m5U, s2U, and Ψ modifications block stimulation of TLR7 and TLR8.
- p. 1 Summary (abstract)
incorporation of modified nucleosides m5C, m6A, m5U, s2U, or pseudouridine ablates activity
Only the authors can say what is right here.
Judge's note: Read p001 image: the Summary sentence is as quoted, naming TLR3, TLR7 and TLR8 immediately before the blanket "ablates activity". Read p003 (crop C006-a shows the same column): "The presence of other nucleoside modifications such as m5C, m5U, Ψ, or m5C/Ψ in the RNA had a less remarkable suppression or no effect at all on the potential of RNA to activate TLR3 (Figure 2B)." Read p004 crop C009-a: "Specifically, m6A and s2U modifications suppress the ability of RNA to stimulate TLR3, whereas m6A, m5C, m5U, s2U, and Ψ modifications block stimulation of TLR7 and TLR8." Read p007 image: "only uridine modifications, such as m5U, s2U, or Ψ, but not m5C or m6A, could abolish the capacity of RNA to activate primary, blood-derived DCs" and "RNA with m6A and s2U modifications did not activate TLR3, and those with m5C, m5U, s2U, m6A, or Ψ did not activate TLR7 or TLR8". Read p009 crop C001-f: "which might explain why Ψ-modified RNA could potently activate TLR3 (Figure 2B)". I looked for a qualifier in the Summary limiting the claim to TLR7/TLR8 or to MDDCs; there is none. The compression in the Summary therefore overstates the receptor-level data the paper reports; rated S3 as a wording/overstatement defect rather than a wrong data value.
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11 minor (typos and the like; most checked by the judge only)
Wording Introduction reads "For example, methylated cytidine in CpG motifs of DNA has proven to be the structural basis of recognition for the innate immune system", whereas the Summary and Discussion state that it is the absence of CpG methylation that is recognised.
- p. 1 Introduction, second paragraph
methylated cytidine in CpG motifs of DNA has proven to be the structural basis of recognition for the innate immune system
- p. 1 Summary
DNA containing methylated CpG motifs, however, is not stimulatory.
- p. 7 Discussion
the lack of such modification in the genomes of microbial pathogens is recognized by TLR9
Only the authors can say what is right here.
Judge's note: Read p001 image: the Introduction sentence is printed exactly as quoted, and two sentences earlier the same paragraph says "secretion of interferon in response to DNA is mediated by unmethylated CpG motifs acting upon TLR9". The Summary on the same page reads "DNA containing methylated CpG motifs, however, is not stimulatory." Read p007 image, Discussion: "In mammalian DNA, cytosines in CpG motifs are mostly methylated, but the lack of such modification in the genomes of microbial pathogens is recognized by TLR9". As printed the Introduction sentence asserts the converse of the paper's other two statements; read loosely as "methylation status is the structural basis of discrimination" it is defensible, so I downgrade to CONFIRMED_MINOR, a wording ambiguity rather than a wrong data claim.
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Typo DC1 is expanded as "primary monocytoid (DC1, BDCA-1+)" in the Results but as "Primary myeloid and plasmacytoid DCs (DC1 and DC2)" in the Experimental Procedures.
- p. 4 Results, right column
we purified primary monocytoid (DC1, BDCA-1+) and plasmacytoid (DC2, BDCA-4+) DCs from peripheral blood
- p. 10 Experimental Procedures, Cells and Cell Culture
Primary myeloid and plasmacytoid DCs (DC1 and DC2) were obtained from peripheral blood
Judge's note: Read p004 crop C009-a: "we purified primary monocytoid (DC1, BDCA-1+) and plasmacytoid (DC2, BDCA-4+) DCs from peripheral blood". Read p010 page image, Cells and Cell Culture: "Primary myeloid and plasmacytoid DCs (DC1 and DC2) were obtained from peripheral blood by using BDCA-1 and BDCA-4 cell isolation kits". The same BDCA-1+ population is given two expansions of the abbreviation DC1 while DC2 is "plasmacytoid" in both places. Both terms are used for BDCA-1+ blood DCs, so the identity of the population is not in doubt; cosmetic inconsistency only.
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Typo Stray full stop before the parenthetical citation: "the relative sensitivity of MDDCs to poly(I):(C) and R-848 treatments also differed. (Figure S1 available in the Supplemental Data with this article online)."
- p. 4 Results, variability paragraph
also differed. (Figure S1 available
Judge's note: Read crop C008-a and crop C010-a (both show this paragraph on p004). The printed text is "...R-848 treatments also differed. (Figure S1 available in the Supplemental Data with this article online). This variability was not observed...". The sentence is closed with a period before the parenthesis, which is then itself closed with a second period, leaving the parenthetical outside the sentence. Punctuation slip only; meaning unaffected.
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Typo Figure 4B row label reads "R848" while the Figure 4 legend and the rest of the paper write "R-848".
- p. 6 Figure 4B, row label
R848
- p. 6 Figure 4 legend
complexed with R-848 (1 g/ml)
Judge's note: Read the p006 page image: the panel B table rows are lipofectin, poly(I):(C), R848, then the RNA series. Read crop C013-b: the legend of the same figure reads "complexed with R-848 (1 µg/ml)", and panel A's third dot plot is headed "R-848". The hyphen is visibly absent in the panel B row and present elsewhere. Cosmetic typographic inconsistency.
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Typo Figure 5 legend misspells adenosine: "Elution times are noted for 3′-monophosphates of pseudouridine (Ψ), cytidine (C), guanosine (G), uridine (U), 7-methylguanosine (m7G), and adenoside (A)."
- p. 7 Figure 5 legend, panel (A)
7-methylguanosine (m7G), and adenoside (A)
- p. 7 Figure 5 legend (A)
adenoside (A)
- p. 7
uridine (U), 7-methylguanosine (m7G), and adenoside (A).
Judge's note: Read crops C014-a and C014-b and the p007 page image. The printed legend reads "...7-methylguanosine (m7G), and adenoside (A)" — the letters are clearly legible in the image, so this is not an extraction artefact. Every other nucleoside in the same list is spelled correctly, and the Methods on p010 and the Introduction on p001 write "N6-methyladenosine" and "adenosine" correctly. Plain misspelling.
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Typo Figure 5 legend punctuation: "the number of m6A, Ψ ,or m5C per molecule of RNA-1571 was calculated" has the comma after the space following Ψ, and the same legend lists "relative to ATP UTP and CTP, respectively" without commas between ATP, UTP and CTP.
- p. 7 Figure 5 legend, panel (B)
the number of m6A, Ψ ,or m5C per molecule of RNA-1571 was calculated
- p. 7 Figure 5 legend (B)
relative to ATP UTP and CTP, respectively
- p. 7 Figure 5 legend (B)
m6A, Ψ ,or m5C per molecule
Judge's note: Read crops C015-a, C015-b, C015-c and the p007 page image. Both slips are visible in the printed legend: "the number of m6A, Ψ ,or m5C per molecule of RNA-1571 was calculated" (the comma is displaced after the space, so this is not an artefact of the Ψ glyph in the text layer), and "“a” represents values (%) for m6ATP, ΨTP, and m5CTP relative to ATP UTP and CTP, respectively" (serial commas present in the first list but absent in the second). Cosmetic punctuation only; meaning unchanged.
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Typo Figure 5 legend, footnote "a": "“a” represents values (%) for m6ATP, ΨTP, and m5CTP relative to ATP UTP and CTP, respectively" — the serial comma after "ATP" is missing in the second list.
- p. 7 Figure 5 legend, footnote "a"
“a” represents values (%) for m6ATP, ΨTP, and m5CTP relative to ATP UTP and CTP, respectively
Judge's note: Read the crop adjudicate/crops/C016-a.png and the full page extract/png/main-p007.png. The printed legend reads "relative to ATP UTP and CTP, respectively" with no punctuation between ATP and UTP, while the parallel first list is punctuated "m6ATP, ΨTP, and m5CTP". Not an extraction artefact; the comma is absent on the page image.
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Typo Discussion, right column: "RIG-1, and PKR that function in the innate immune system independently from TLRs (Sen and Sarkar, 2005)" — the sensor is printed as "RIG-1" with the digit 1.
- p. 9 Discussion, right column
RIG-1, and PKR that function in the innate immune system independently from TLRs
- p. 9 Discussion, last paragraph
RIG-1, and PKR
Only the authors can say what is right here.
Judge's note: Rests on outside knowledge: the cytoplasmic RNA helicase is named RIG-I (retinoic acid-inducible gene I, Roman numeral I). On the page image extract/png/main-p009.png and crops adjudicate/crops/C018-a.png and C018-b.png the glyph after the hyphen is clearly the digit 1 (same form as the "1" in "DC1", "TLR3-293" numerals on the page) and unlike the capital I of "IL-8" / "IRB" in the same column. Both reviewer notes point at the same single occurrence, which continues the sentence begun at the foot of the left column ("cytoplasmic receptors such as FADD,"). Cosmetic name misspelling.
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Typo Experimental Procedures, Cells and Cell Culture: "Leukopheresis samples were obtained from HIV-uninfected volunteers through an IRB-approved protocol."
- p. 9 Experimental Procedures, Cells and Cell Culture
Leukopheresis samples were obtained from HIV-uninfected volunteers
Only the authors can say what is right here.
Judge's note: Rests on outside knowledge: the standard form of the word is "leukapheresis" (from apheresis). Verified the printed spelling "Leukopheresis" on the full page image extract/png/main-p009.png (Cells and Cell Culture, right column), so it is not an extraction artefact. Rated minor because "leukopheresis" is a variant that appears in the older clinical literature, so only the authors/copyeditor can say whether it should be changed.
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Typo Experimental Procedures, Natural and Synthetic RNA: "RNA was isolated from the purified mitochondria, cytoplasmic and nuclear fractions of 293 cells, unfractioned 293 cells, rat liver, mouse cell line TUBO..." — "unfractioned" for "unfractionated".
- p. 10 Experimental Procedures, Natural and Synthetic RNA
nuclear fractions of 293 cells, unfractioned 293 cells, rat liver
Judge's note: Verified on crop adjudicate/crops/C021-a.png and on the full page extract/png/main-p010.png; the word is printed "unfractioned", contrasted with the cytoplasmic and nuclear fractions named immediately before. Not an extraction artefact; cosmetic misspelling with an evident fix.
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Typo The same supplier is printed two ways on PDF page 10: "6 mM m7GpppG cap analog (New England BioLabs, Beverly, MA)" (In Vitro-Transcribed RNA) and "Aliquots of RNA-730 were digested with alkaline phosphatase (New England Biolab)" (Natural and Synthetic RNA).
- p. 10 Natural and Synthetic RNA
New England Biolab)
- p. 10 In Vitro-Transcribed RNA
New England BioLabs, Beverly, MA
Judge's note: Both forms verified on the full page image extract/png/main-p010.png and the second on crops adjudicate/crops/C021-a.png and C022-a.png; the left column shows "(New England BioLabs, Beverly, MA)" in the transcription paragraph and "(New England Biolab)" in the phosphatase sentence lower in the same column. The internal inconsistency is established from the file itself; that the registered name is "New England Biolabs" (plural) rests on outside knowledge. Cosmetic.
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