Duplexes of 21-Nucleotide RNAs Mediate RNA Interference in Cultured Mammalian Cells
2001 · paper · Biomedicine
The text provided contains only the methods and reference list of a different Nature letter on B-cell antigen receptors and antigen presentation. It does not show the paper's findings or its stated subject.
4 findings
Substance The Fig. 3 legend states "(43-nucleotide uninterrupted identity in 49-bp segment, 239-nucleotide longest uninterrupted identity in 484-bp segment)", but with the paper's own coordinates the longest uninterrupted GL2/GL3 identity inside the 49-bp segment can be at most 41 nucleotides.
- p. 3 Figure 3 legend, final sentence
(43-nucleotide uninterrupted identity in 49-bp segment, 239-nucleotide longest uninterrupted identity in 484-bp segment)
- p. 4 Methods, RNA preparation
The 49- and 484-bp GL2 or GL3 dsRNAs corresponded to positions 113–161 and 113–596, respectively, relative to the start of translation
- p. 2 Figure 1b, GL2 and GL3 siRNA sequences
5'-CGUACGCGGAAUACUUCGATT
- p. 2 Figure 1b, GL3 siRNA sequence and legend
The GL2 and GL3 siRNA duplexes differ by only three single-nucleotide substitutions (boxed in grey).
Only the authors can say what is right here.
Judge's note: Legend read on the page 3 image; Methods read on the page 4 image ('The 49- and 484-bp GL2 or GL3 dsRNAs corresponded to positions 113-161 and 113-596 ... relative to the start of translation'; 'siRNA sequences targeting GL2 ... and GL3 luciferase corresponded to the coding regions 153-173'). Crop C007-c and the page-2 text layer give the sense strands GL2 5'-CGUACGCGGAAUACUUCGATT and GL3 5'-CUUACGCUGAGUACUUCGATT; comparing them position by position I find exactly three differences, at duplex positions 2, 8 and 11 (matching the three grey boxes and the Fig. 1 legend's 'three single-nucleotide substitutions'). With position 1 = coding position 153, these are coding positions 154, 160 and 163. Two of them (154, 160) lie inside 113-161, so the identity runs inside that segment are 113-153 = 41 nt, 155-159 = 5 nt and 161 = 1 nt; the maximum is 41, and any further GL2/GL3 differences in 113-152 would only shorten it. 43 is therefore not reachable. Either the legend's '43' or one of the coordinate sets is off; only the authors can say which.
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Reference or label The sentence "In Drosophila S2 cells (Fig. 2a and b), the specific inhibition of luciferases was complete and similar to results previously obtained for longer dsRNAs" carries superscript "6,10,12,19", but ref. 19 is "Clemens, M. & Williams, B. Inhibition of cell-free protein synthesis by pppA2'p5'A2'p5'A: a novel oligonucleotide synthesized by interferon-treated HeLa cells. Cell 13, 565-572 (1978)".
- p. 2 p.495 left column, paragraph on S2 cells
previously obtained for longer dsRNAs6,10,12,19
- p. 5 reference list, ref. 19
Inhibition of cell-free protein synthesis by pppA
- p. 5 ref. 19
Inhibition of cell-free protein synthesis by pppA2'p5'A2'p5'A
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Judge's note: Superscript read on the page 2 image and confirmed in the intact page-2 text layer (extract/main.layout.txt line 171: 'for longer dsRNAs6,10,12,19'). Ref. 19 read on the page 5 image: it is the 1978 2-5A/cell-free protein synthesis paper, whose printed title is unrelated to RNAi in Drosophila S2 cells with long dsRNA. Refs 6, 10 and 12 in the same superscript are Drosophila-cell RNAi papers; ref. 11 (Clemens, J. C. et al., 'Use of double-stranded RNA interference in Drosophila cell lines'), which would fit, is not in this superscript although it is cited in the Introduction. Ref. 19 is cited nowhere else in the article (I checked the interferon paragraph on page 2, where the ribonuclease L sentence carries no superscript). Which citation was intended is for the authors.
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Reference or label The gene-selection sentence "lamin A/C, lamin B1, nuclear mitotic apparatus protein (NuMA) and vimentin" carries superscript 27, which is "Razin, A. CpG methylation, chromatin structure and gene silencing-a three-way connection. EMBO J. 17, 4905-4908 (1998)", not the cytoskeleton guidebook (ref. 22, Kreis & Vale).
- p. 3 p.496 right column, endogenous-gene paragraph
nuclear mitotic apparatus protein (NuMA) and vimentin27
- p. 5 reference list, ref. 27
Razin, A. CpG methylation, chromatin structure and gene silencing
- p. 3 Results, endogenous genes paragraph
nuclear mitotic apparatus protein (NuMA) and vimentin
- p. 5 Reference list, ref. 22
Guidebook to the Cytoskeletal and Motor Proteins
Only the authors can say what is right here.
Judge's note: Read the superscript as 27 on the page 3 image (the page-3 text layer is OCR and renders it only as '?'). Checked the reference list on the page 5 image: ref. 27 is the Razin CpG-methylation review; ref. 22 is 'Kreis, T. & Vale, R. Guidebook to the Cytoskeletal and Motor Proteins, Parts 2b and 3a'. Internal evidence supports the slip: the journal numbers references in order of first citation, and in this article refs 21 (hG/pAD3, page 3) and 23 (lamin A/C knock-out mice, page 4 image) bracket this sentence, with refs 24-27 all first cited in the Discussion methylation paragraph on page 4 ('nucleotide siRNAs24', 'transcriptional silencing25', 'does not26', 'well documented processes27'). Ref. 22 is cited nowhere else in the text, so the vimentin sentence is the only slot it can occupy. The authors should confirm the intended number.
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Reference or label Reference 3 prints an impossible page range: "Hammond, S. M., Caudy, A. A. & Hannon, G. J. Post-transcriptional gene silencing by double-stranded RNA. Nature Rev. Genet. 2, 110-1119 (2001)."
- p. 5 Reference list, ref. 3
Nature Rev. Genet. 2, 110–1119 (2001).
- p. 5 Reference list, ref. 3
Nature Rev. Genet. 2, 110–1119 (2001)
Judge's note: Read the entry on the page 5 image; the four digits '1119' follow the dash in the rendered page, so this is not an extraction artefact. An article starting at page 110 and ending at page 1119 would run 1,010 pages, which is not credible for a review; the end page carries a duplicated digit. I confirm only the internal implausibility of the printed range as the defect.
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6 minor (typos and the like; most checked by the judge only)
Wording The Introduction cites refs 10,12 for the claim that "it has not been possible to detect potent and specific RNA interference" in mammalian lines "including ... CHO-K1 (Chinese hamster ovary) cells, applying dsRNA that varies in size between 38 and 1,662 base pairs (bp)", while the Discussion says of the same ref. 12 "Specific silencing effects with dsRNAs (356-1,662 bp) were previously reported in CHO-K1 cells"; the 38-bp lower bound is not supported by the dsRNA lengths the paper itself attributes to refs 10 and 12.
- p. 1 Introduction, first body paragraph, right column
and CHO-K1 (Chinese hamster ovary) cells, applying dsRNA that varies in size between 38 and 1,662 base pairs (bp)
- p. 3 Discussion, left column (long-dsRNA paragraph)
Specific silencing effects with dsRNAs (356–1,662 bp) were previously reported in CHO-K1 cells
- p. 5 Reference 12
Ui-Tei, K., Zenno, S., Miyata, Y. & Saigo, K. Sensitive assay of RNA interference in Drosophila and Chinese hamster cultured cells
Only the authors can say what is right here.
Judge's note: Read page 1 image (right column, first body paragraph) and page 3 image (Discussion, left column). Both quotes are present as given. The tension over whether ref. 12 showed specific silencing in CHO-K1 is largely defused by the word 'potent' and by the Discussion's own qualifier ('the amounts of dsRNA required to detect a 2- to 4-fold specific reduction were about 20-fold higher than in our experiments'), so I downgrade that part. What remains unexplained internally is the length range: the paper attributes 356-1,662 bp to ref. 12 (CHO-K1) and 829-bp lacZ / 717-bp GFP to ref. 10 (293, NIH/3T3, BHK-21), none of which supports a 38-bp lower bound for the mammalian experiments described in that sentence. Wording/ambiguity only.
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Wording The text states "Sequence-specific silencing by 50- or 500-bp dsRNAs reduced the targeted reporter-gene expression by an additional 2- to 5-fold", but two of the targeted bars in Fig. 3c sit near 0.6 of the hG control, i.e. about a 1.6-fold reduction, below the stated lower bound.
- p. 3 Discussion, left column
Sequence-specific silencing by 50- or 500-bp dsRNAs reduced the targeted reporter-gene expression by an additional 2- to 5-fold.
- p. 3 Figure 3c (normalized ratios, hG-normalized groups)
Ratios of normalized target to control luciferase.
Only the authors can say what is right here.
Judge's note: Read the sentence and Fig. 3c on the page 3 image. In the hG-normalized groups the black hG bars are set to 1; the targeted bars read approximately 0.18 (GL2, 50 bp) and 0.62 (RL, 50 bp) in the 50-bp group, and approximately 0.62 (GL2, 484 bp) and 0.20 (RL, 501 bp) in the 500-bp group. Two of these correspond to roughly 1.6-fold, and the smallest corresponds to roughly 5.6-fold, so the printed '2- to 5-fold' range does not cover the plotted values at either end. Values are read off a linear 0-1.2 axis, so the reading carries some uncertainty, and the text adds that 'Similar effects were also detected in the other three mammalian cell lines tested (data not shown)', which the range may partly summarise; I therefore treat this as an overstated/imprecise description rather than a hard numerical error.
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Wording In one Methods paragraph describing "21-nucleotide RNAs", the reporter siRNA targets are given as 21-nucleotide spans ("coding regions 153-173"; "region 119-139") while the four endogenous-gene siRNA targets are given as 23-nucleotide spans ("position 608-630"; "from position 672-694"; "from position 3,988-4,010"; "from position 346-368"), with no statement of the change of convention.
- p. 4 Methods, RNA preparation
corresponded to the coding regions 153–173 relative to the first nucleotide of the start codon; siRNAs targeting RL (Acc. No. AF025846) corresponded to region 119–139
- p. 4 Methods, RNA preparation
was from position 608–630 relative to the start codon; lamin B1 (Acc. No. NM_005573) siRNA was from position 672–694; NuMA (Acc. No. Z11583) siRNA from position 3,988–4,010, and vimentin (Acc. No. NM_003380) from position 346–368
- p. 4 Methods, RNA preparation, opening sentence
21-nucleotide RNAs were chemically synthesized
Only the authors can say what is right here.
Judge's note: All six coordinate pairs read from the page 4 image (Methods, RNA preparation), together with the opening '21-nucleotide RNAs were chemically synthesized'. Inclusive counting gives 21 for the two luciferase spans and 23 for all four endogenous-gene spans. The same paragraph's dsRNA spans (113-161 = 49 bp, 113-596 = 484 bp, 118-167 = 50 bp, 118-618 = 501 bp, 121-170 = 50 bp, 121-621 = 501 bp) all check out under inclusive counting, confirming that convention. A 23-nt span could legitimately quote the region covered by a 21-mer duplex including the two positions opposite the dTdT overhang, but nothing in the article or Methods says so, and the reporter spans in the same paragraph use the other convention. Unresolved ambiguity rather than a demonstrable wrong number.
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Wording "Lamin A and C are produced by alternative splicing in the 3' region and are present in equal amounts in the lamina of mammalian cells (Fig. 4j, k)" points at panel k, which the legend defines as the vimentin blot ("j, k, Western blots of transfected cells using lamin A/C- (j) or vimentin-specific (k) antibodies").
- p. 4 Main text, first paragraph of left column
are present in equal amounts in the lamina of mammalian cells (Fig. 4j, k).
- p. 4 Figure 4 legend
j, k, Western blots of transfected cells using lamin A/C- (j) or vimentin-specific (k) antibodies.
- p. 4 Discussion, left column
are present in equal amounts in the lamina of mammalian cells (Fig. 4j, k)
Judge's note: Read the sentence and the Fig. 4 legend on the page 4 image, and looked at the figure itself: panel j shows the lamin A and lamin C bands, panel k shows a single vimentin band used as the loading control. The relative abundance of lamin A versus lamin C is visible only in panel j, and both bands lie in the same lane, so the loading control adds nothing to that particular comparison. Citing the two western-blot panels jointly is a defensible shorthand (they are one stripped and re-probed blot, and the earlier 'Fig. 4j, k' for the >90% reduction is appropriate), so this is a loose pointer rather than a wrong one.
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Typo The results text reads "In NIH/3T3, monkey COS-7 and Hela S3 cells (Fig. 2c-h)", using 'Hela' where the figure panels, figure legend and Methods all print 'HeLa S3'.
- p. 2 Right column, second paragraph of results
In NIH/3T3, monkey COS-7 and Hela S3 cells (Fig. 2c–h)
- p. 2 Figure 2, panel titles g and h
HeLa S3
- p. 4 Methods, Cell culture
293, NIH/3T3, HeLa S3, HeLa SS6, COS-7 cells
- p. 2 Results, first column
monkey COS-7 and Hela S3 cells (Fig. 2c–h)
Judge's note: Read on the page 2 image and confirmed in the intact page-2 text layer (extract/main.layout.txt line 175: 'COS-7 and Hela S3 cells (Fig. 2c±h)'). Crop C004-b shows the Fig. 2g,h panel titles printed 'HeLa S3', and the page 4 image shows 'HeLa S3, HeLa SS6' in Cell culture. Purely a capitalisation inconsistency.
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Typo "As a control for nonspecific inhibition, dsRNAs from humanized GFP (hG) was used." - plural subject with singular verb.
- p. 3 Discussion, left column (long-dsRNA paragraph)
As a control for nonspecific inhibition, dsRNAs from humanized GFP (hG)21 was used.
Judge's note: Read on the page 3 image (left column, long-dsRNA paragraph); the OCR text layer agrees. Two hG dsRNAs are in fact used (Methods on the page 4 image: '50- and 501-bp hG dsRNA corresponded to positions 121-170 and 121-621'; Fig. 3 legend names 'hG' controls at both lengths), so the plural subject is intended and 'was' should be 'were'. Cosmetic grammar only.
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