RNA regulons in Hox 5' UTRs confer ribosome specificity to gene regulation

doi:10.1038/nature14010

. 2015 Jan 1;517(7532):33-8.

doi: 10.1038/nature14010. Epub 2014 Nov 19.

RNA regulons in Hox 5' UTRs confer ribosome specificity to gene regulation

Shifeng Xue¹, Siqi Tian², Kotaro Fujii³, Wipapat Kladwang², Rhiju Das⁴, Maria Barna³

Affiliations

¹ 1] Department of Developmental Biology, Stanford University, Stanford, California 94305, USA [2] Department of Genetics, Stanford University, Stanford, California 94305, USA [3] Tetrad Graduate Program, University of California, San Francisco, San Francisco, California 94158, USA.
² Department of Biochemistry, Stanford University, Stanford, California 94305, USA.
³ 1] Department of Developmental Biology, Stanford University, Stanford, California 94305, USA [2] Department of Genetics, Stanford University, Stanford, California 94305, USA.
⁴ 1] Department of Biochemistry, Stanford University, Stanford, California 94305, USA [2] Department of Physics, Stanford University, Stanford, California 94305, USA.

PMID: 25409156
PMCID: PMC4353651
DOI: 10.1038/nature14010

RNA regulons in Hox 5' UTRs confer ribosome specificity to gene regulation

Shifeng Xue et al. Nature. 2015.

. 2015 Jan 1;517(7532):33-8.

doi: 10.1038/nature14010. Epub 2014 Nov 19.

Authors

Shifeng Xue¹, Siqi Tian², Kotaro Fujii³, Wipapat Kladwang², Rhiju Das⁴, Maria Barna³

Affiliations

¹ 1] Department of Developmental Biology, Stanford University, Stanford, California 94305, USA [2] Department of Genetics, Stanford University, Stanford, California 94305, USA [3] Tetrad Graduate Program, University of California, San Francisco, San Francisco, California 94158, USA.
² Department of Biochemistry, Stanford University, Stanford, California 94305, USA.
³ 1] Department of Developmental Biology, Stanford University, Stanford, California 94305, USA [2] Department of Genetics, Stanford University, Stanford, California 94305, USA.
⁴ 1] Department of Biochemistry, Stanford University, Stanford, California 94305, USA [2] Department of Physics, Stanford University, Stanford, California 94305, USA.

PMID: 25409156
PMCID: PMC4353651
DOI: 10.1038/nature14010

Abstract

Emerging evidence suggests that the ribosome has a regulatory function in directing how the genome is translated in time and space. However, how this regulation is encoded in the messenger RNA sequence remains largely unknown. Here we uncover unique RNA regulons embedded in homeobox (Hox) 5' untranslated regions (UTRs) that confer ribosome-mediated control of gene expression. These structured RNA elements, resembling viral internal ribosome entry sites (IRESs), are found in subsets of Hox mRNAs. They facilitate ribosome recruitment and require the ribosomal protein RPL38 for their activity. Despite numerous layers of Hox gene regulation, these IRES elements are essential for converting Hox transcripts into proteins to pattern the mammalian body plan. This specialized mode of IRES-dependent translation is enabled by an additional regulatory element that we term the translation inhibitory element (TIE), which blocks cap-dependent translation of transcripts. Together, these data uncover a new paradigm for ribosome-mediated control of gene expression and organismal development.

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing interests.

Figures

**Extended Data Figure 1. HoxA IRES controls confirming that Fluc activity from bicistronic vector is due to IRES activity**
a, qPCR of both Rluc and Fluc from transfected cells shows that Rluc and Fluc are produced at the same ratio. All Rluc and Fluc values are normalized to that of HCV (set to 1). n = 3; individual experiments performed in duplicates. **b–c,** shRNA against Rluc decreased reporter activity of both Rluc (a) and Fluc (b), confirming that Rluc and Fluc were transcribed on the same mRNA. n= 3 individual experiments performed in triplicates. d, RT-PCR using primers in Rluc and Fluc show that there is no cryptic splice site in the cloned Hox 5′UTR. Primer locations are shown as arrows in the diagram. e, Inserting a strong hairpin (−67 kcal/mol) after the Rluc reporter did not affect Fluc activity, suggesting Fluc activity was not due to ribosome readthrough.

**Extended Data Figure 2. Disruption of RPL38 in C3H10T1/2 by TALEN nucleases**
a, Location of TALEN pairs. 2 pairs of TALENs were designed to bind at the end of Exon 2 and the beginning of Exon 3 to make a genomic break close to the ATG. Sequencing of a positive clone shows a deletion of the ATG and most of the intron after it. Coding sequence is highlighted in green. b RPL38 knock down does not change cap-dependent translation (Rluc) but decreases IRES-dependent translation (Fluc) from specific Hox 5′UTRs. Luciferase activity was normalized to amount of Fluc RNA in the cells as quantified by qPCR. *p< 0.05 (t-test compared to Ctrl). n = 2 individual experiments performed in duplicates.

**Extended Data Figure 3. Alignment of *Hoxa9* IRES element between vertebrate species**
Nucleotides 944 to 1266 of the mouse *Hoxa9* 5′UTR were aligned with sequences from other vertebrates and show high sequence homology. Nucleotides are colored based on their homology.

**Extended Data Figure 4. Chemical mapping and secondary structure prediction of full-length *Hoxa9* 5′UTR**
a, Secondary structure prediction of full-length *Hoxa9* using ligSHAPE data. The *Hoxa9* IRES element (nt 957–1132, shaded in green) is predicted as the same secondary structure shown in Fig. 2a. b, Normalized SHAPE reactivity of *Hoxa9* IRES (nt 957–1132 and 944–1266 from CE-based 1-dimensional SHAPE, full-length 1–1266 from MiSeq-based ligSHAPE). c, Normalized SHAPE reactivity of *Hoxa9* TIE (nt 1–342 from CE-based 1-dimensional SHAPE, full-length 1–1266 from MiSeq-based ligSHAPE).

**Extended Data Figure 5. Chemical mapping and secondary structure prediction of full length *Hoxa5* 5′UTR**
a, Secondary structure prediction of *Hoxa5* using 1-dimensional SHAPE data. Nucleotides are colored with SHAPE reactivities. Percentage labels give bootstrap support values for each helix. The feature highlighted in blue resembles P3 in *Hoxa9* and the tip highlighted in pink is deleted in **b. b**, The deletion of the tip identified in *Hoxa5* IRES structure shown in a decreases IRES activity in bicistronic reporter assays. IRES activity was normalized to full length *Hoxa5* 5′UTR (A5, set to 1). **p< 0.01 (t-test as compared to A5). n= 2 experiments; performed in triplicates. c, Both Hoxa9 and Hoxa5 contain an asymmetric bulge in a region important for IRES activity. **d-e**, Normalized SHAPE (d) and DMS (e) reactivity of *Hoxa5* (CE-based and MiSeq-based).

**Extended Data Figure 6. Secondary structure prediction and mutate-and-map (M²) dataset of *Hoxa9* IRES element**
**a–b**, Entire M² dataset and Z-score contact-map of *Hoxa9* nt 957–1132 across 177 single mutants probed by 1M7. c, Secondary structure prediction of *Hoxa9* nucleotides 957–1132 using M² data alone. The model contains the same helices as the model from combined SHAPE/ M² analysis in Fig. 2a, up to register shifts and edge base pairs; the small rearrangements are labeled P3b′, P3c′, P3d, P4b′ and P4c′. d, Secondary structure of *Hoxa9* nt 957–1132 using 1-dimensional SHAPE data alone. Nucleotides are colored with SHAPE reactivity. The model contains the same helices as the model from combined SHAPE/M² analysis in Fig. 2a, up to register shifts and edge base pairs; the small rearrangements are labeled P3b′, P3c′, and P4c. e, Secondary structure prediction and bootstrap support matrix of *Hoxa9* nt 944–1266 using 1-dimensional SHAPE data. The model agrees with the model derived from SHAPE analysis of the 957–1132 subdomain in Fig. 2a.

**Extended Data Figure 7. Mutation/rescue results of *Hoxa9* IRES structure (nt 944–1266) probed by 1M7**
Electropherograms of mutation/rescue to test base-pairings in P3c (**a–b**), P3b (**c–e**), P3a (**f–k**), P4b (**l–o**), P4a (**p–u**) and pk3-4 (**v–ai**). Perturbation of the chemical mapping reactivities by mutations of one strand and restoration by mutations in the other strand provide strong evidence for the tested pairings in P3c (**a–b**), P3b (**c–e**), P3a (**f–k**), P4b (**m–o**) and P4a (**p–q**). Lack of rescue in other tested pairings is consistent with either absence of those pairings or higher-order structure (*e.g.,* base triples) interacting with those pairings.

**Extended Data Figure 8. Putative uORFs within the 5′UTRs of *Hoxa9* and *Hoxa5* do not inhibit cap-dependent translation and *Hoxa9*^Δ*^IRES* targeting strategy**
uORFs are marked by black circles on the diagram of monocistronic reporter for the (a) *Hoxa9* and (b) *Hoxa5* 5′UTR. All the ATGs in the 5′UTR were mutated to TTG in A9ΔuORF construct and GTG in A5ΔuORF. The IRES element (944-1266) was removed in A9 IRES construct. The IRES element was removed from the A9ΔuORF construct in A9ΔIRESΔuORF. n= 3 individual experiments in duplicates. Data represent mean ± s.d. c, Diagrams of the *Hoxa9* locus and the targeting vector. Boxes represent exons, grey boxes represent UTRs and black boxes represent the coding sequence. Nucleotides 944-1145 were replaced by a floxed Neo cassette in the targeting vector. Locations of Southern blot probes, restriction enzymes used for Southern analysis and expected sizes are marked on the diagrams. d, Southern blot analysis of targeted cells using both the 5′ and 3′ probes showing that both arms integrated correctly into the *Hoxa9* locus.

**Extended Data Figure 9. The presence of Neo cassette in the *Hoxa9* locus is linked to the presence of an L1 →T13 homeotic transformation**
a, Diagram of the *Hoxa9* locus (top) and axial skeleton phenotype (bottom) in different *Hoxa9* mouse mutants. The original *Hoxa9^−/−* was made by replacing the homeodomain with a Neo cassette. Vertebra with homeotic transformation is colored red. b, Representative skeletons of *Hoxa9*^+/+, *Hoxa9^Neo/+* and *Hoxa9^Neo/Neo*. Arrows point to the additional rib(s) on L1, revealing a homeotic transformation to T13. These results show that it is the presence of Neo in the targeting locus, which may affect the expression of neighboring Hox gene, that is sufficient to cause the L1 → T13 phenotype. When the Neo cassette is removed from the targeting locus by crossing the *Hoxa9^Neo/+* mouse with a CMV Cre line, the L1 → T13 phenotype is no longer present. n = 3 embryos of each genotype.

**Extended Data Figure 10. Sucrose gradient fractionation shows no difference in *β-actin* association with polysomes in *Hoxa9*^+/+ and *Hoxa9*^Δ*^IRES/*^Δ*^IRES* embryos**
a, Overlay of A₂₆₀ trace during fractionation showing no difference in polysome profiles between E11.5 *Hoxa9*^+/+ and *Hoxa9*^Δ*^IRES/*^Δ*^IRES* embryos. b, qPCR from each fraction reveals no difference in *B-actin* mRNA accumulation between *Hoxa9*^+/+ and *Hoxa9*^Δ*^IRES/*^Δ*^IRES* embryos. c, Quantification of *B-actin* mRNA in fractions. Fractions 1-8 are pre-polysomes and 9-16 are polysome fractions. n = 3 embryos of each genotype.

**Figure 1. Select HoxA 5′UTRs contain IRES elements regulated by RPL38**
a, HoxA genes are located in tandem along a chromosomal locus, and their location directs their anterior expression boundaries along the embryo. Striped boxes, Hox genes translationally regulated by RPL38. IRES activity is the ratio between Fluc and Rluc. b, IRES activity of HoxA 5′UTRs. IRES activity of UTRs was normalized to that of empty vector (pRF) and RPL38-regulated Hox genes are denoted in red. ** p< 0.01 (t-test as compared to pRF). n= 5. c, RPL38 regulates translation of specific HoxA IRES elements. Bicistronic reporters were transfected into control cells (CTRL) or cells where one copy of Rpl38 was deleted (RPL38 KD). Top: Western blot of RPL38 and β-actin with quantification of RPL38 levels below blot. Bottom: IRES activity of specific Hox 5′UTRs in RPL38 KD cells normalized to that of control cells. ** p< 0.01 (t-test). n= 3. d, Deletion analysis of the Hoxa9 5′UTR. IRES activity was normalized to that of the full-length Hoxa9 5′UTR (A9). AS: anti-sense. **p<0.01 (t-test as compared to pRF). n= 3. e, IRES activity of the *Hoxa9* IRES (nt 944–1266) in RPL38 KD cells. **p<0.01 (t-test). n= 3. f, Alignment of the Hoxa9 5′UTR in vertebrates. g, The 5′UTR of one of two zebrafish *Hoxa9* paralogs (*Hoxa9b*) has similar IRES activity as the mouse *Hoxa9* IRES. IRES activity was normalized to pRF. **p< 0.01 (t-test as compared to pRF). n= 3. All experiments were performed in triplicates. Data represent mean ± s.d.

**Figure 2. The *Hoxa9* IRES forms a stable RNA structure that facilitates recruitment of the ribosome**
a, Secondary structure of *Hoxa9* 5′UTR (nt 957–1132) by SHAPE and mutate-and-map analysis. Nucleotides are colored with SHAPE reactivities. Percentages give bootstrap support values for each helix, including a tentative pseudoknot (pk). Base-pairs verified by mutation and rescue are circled green. Inset: mutations made in helix P3a for functional testing in **b. b,** Deletions as well as mutation-and-rescue of specific hairpins identified in *Hoxa9* IRES structure shown in a affects IRES activity in bicistronic reporter assays. IRES activity was normalized to full-length *Hoxa9* 5′UTR (A9, set to 1). **p< 0.01 (t-test as compared to A9). n= 3 experiments performed in triplicates. c, Schematic of RNA pull-down experiments. d, e, Representative western blot for RPs (d) and qPCR for rRNA (e) from elutes of two independent RNA pull-down experiments. The 18S and 28S rRNA amounts of all *Hoxa9* deletion mutants were normalized to amount of full-length *Hoxa9* 5′UTR (set to 1) eluted from beads. n= 2. Data represent mean ± s.d.

**Figure 3. Identification of minimal IRES domains in all IRES-containing HoxA 5′UTRs**
Deletion analysis of HoxA 5′UTRs performed in bicistronic constructs maps the IRES element in *Hoxa3* (a), *Hoxa4* (b), *Hoxa5* (c) and *Hoxa11* (d). IRES activity was normalized to full-length 5′UTRs (FL). ** p< 0.01 (t-test compared to pRF). n= 3 experiments performed in triplicates. Data represent mean ± s.d.

**Figure 4. Identification of the TIE that blocks cap-dependent translation**
a, Deletions within the *Hoxa9* 5′UTR in a monocistronic reporter assay. Fluc activity was normalized to amount of luciferase mRNA determined by qPCR. All values are normalized to Hoxa9 full-length 5′UTR (A9), set to 1. n= 4. b, A TIE that inhibits cap-dependent translation was identified in the *Hoxa9* 5′UTR (nt 1–342). n= 3. **c-e,** Deletion of the minimal IRES elements within monocistronic reporters of HoxA 5′UTRs and identification of TIEs close to the mRNA cap in *Hoxa3* (c), *Hoxa4* (d) and *Hoxa5* (e). All values are normalized to the full-length constructs (FL). n= 3. f, *HoxA* TIEs were placed upstream of the β-globin 5′UTR (Hbb) in a monocistronic reporter to assay inhibition of cap-dependent translation. n= 3. All experiments were performed in duplicates. Data represent mean ± s.d. **p< 0.01 (t-test).

**Figure 5. The *Hoxa9* IRES is required for axial skeleton patterning but not for *Hoxa9* mRNA expression**
a, Scheme of *Hoxa9* IRES targeted deletion in the mouse. LoxP sites are depicted by yellow triangles. The region deleted is highlighted in orange. b, Representative *in situ* hybridization of *Hoxa9* mRNA expression at E11.5. n = 3 embryos of each genotype. Arrows indicate the anterior boundaries of expression in the neural tube and somites. c, qPCR shows little change in *Hoxa9* transcript levels in neural tube and somites of *Hoxa9*^Δ*^IRES/+* and *Hoxa9*^Δ*^IRES/*^Δ*^IRES* embryos. *Hoxa9*^Δ*^IRES/+,* p = 0.06; *Hoxa9*^Δ*^IRES/*^Δ*^IRES* p = 0.03, t-test as compared to *Hoxa9*^+/+. n ≥ 4 embryos of each genotype. d, Representative skeletons of *Hoxa9*^+/+, *Hoxa9*^Δ*^IRES/+* and *Hoxa9*^Δ*^IRES/*^Δ*^IRES* mice. Arrows point to the missing rib(s) on T13, normally present in WT mice (arrowheads) revealing a posterior homeotic transformation to L1. n = 5 mice of each genotype.

Figure 6. The *Hoxa9* IRES is critically required for HOXA9 translation *in vivo*
a, Representative immunostaining of HOXA9 (green) in cross-section of E11.5 *Hoxa9^+/+* (top) and *Hoxa9*^Δ*^IRES/*^Δ*^IRES* (bottom) embryos from the thoracic to sacral levels. DAPI staining is in blue. NT: neural tube, som: somite. n = 3 embryos of each genotype. b, Somites and neural tubes of E11.5 *Hoxa9*^+/+ and *Hoxa9*^Δ*^IRES/*^Δ*^IRES* embryos were microdissected and fractionated on a sucrose gradient. *Middle:* qPCR of *Hoxa9* mRNA from each fraction. Early pre-polysome fractions are highlighted. *Right*: quantification of *Hoxa9* mRNA in fractions. Fractions 1–8 are pre-polysome and 9–16 polysome fractions. **p< 0.01 (t-test compared to *Hoxa9*^+/+). n = 3 embryos of each genotype. c, Model for how ribosome-mediated regulation of gene expression is encoded within mRNA sequence. Stress-responsive cellular IRES elements are only active when cap-dependent translation is down-regulated through a decrease in eIF4F activity. HoxA mRNAs possess a TIE, which normally inhibits cap-dependent translation. The TIE enables ribosome-mediated control of HoxA IRES expression during embryonic development.

See this image and copyright information in PMC

Comment in

Molecular biology: Entry signals control development.
Dinman JD. Dinman JD. Nature. 2015 Jan 1;517(7532):24-5. doi: 10.1038/nature14069. Epub 2014 Nov 19. Nature. 2015. PMID: 25409148 Free PMC article.

Cited by

False-positive IRESes from Hoxa9 and other genes resulting from errors in mammalian 5' UTR annotations.
Akirtava C, May GE, McManus CJ. Akirtava C, et al. Proc Natl Acad Sci U S A. 2022 Sep 6;119(36):e2122170119. doi: 10.1073/pnas.2122170119. Epub 2022 Aug 29. Proc Natl Acad Sci U S A. 2022. PMID: 36037358 Free PMC article.
Are there roles for heterogeneous ribosomes during sleep in the rodent brain?
Buchanan IM, Smith TM, Gerber AP, Seibt J. Buchanan IM, et al. Front Mol Biosci. 2022 Oct 6;9:1008921. doi: 10.3389/fmolb.2022.1008921. eCollection 2022. Front Mol Biosci. 2022. PMID: 36275625 Free PMC article.
Designing synthetic RNAs to determine the relevance of structural motifs in picornavirus IRES elements.
Fernandez-Chamorro J, Lozano G, Garcia-Martin JA, Ramajo J, Dotu I, Clote P, Martinez-Salas E. Fernandez-Chamorro J, et al. Sci Rep. 2016 Apr 7;6:24243. doi: 10.1038/srep24243. Sci Rep. 2016. PMID: 27053355 Free PMC article.
Analysis of the 5' Untranslated Region Length-Dependent Control of Gene Expression in Maize: A Case Study with the ZmLAZ1 Gene Family.
Liu B, Liu X, Sun M, Sun Y, Liu D, Hao L, Tao Y. Liu B, et al. Genes (Basel). 2024 Jul 29;15(8):994. doi: 10.3390/genes15080994. Genes (Basel). 2024. PMID: 39202355 Free PMC article.
The m⁶A epitranscriptome: transcriptome plasticity in brain development and function.
Livneh I, Moshitch-Moshkovitz S, Amariglio N, Rechavi G, Dominissini D. Livneh I, et al. Nat Rev Neurosci. 2020 Jan;21(1):36-51. doi: 10.1038/s41583-019-0244-z. Epub 2019 Dec 5. Nat Rev Neurosci. 2020. PMID: 31804615 Review.

See all "Cited by" articles

References

1. Kondrashov N, et al. Ribosome-Mediated Specificity in Hox mRNA Translation and Vertebrate Tissue Patterning. Cell. 2011;145:383–397. - PMC - PubMed
1. Lee A, Burdeinick-Kerr R, Whelan S. A ribosome-specialized translation initiation pathway is required for cap-dependent translation of vesicular stomatitis virus mRNAs. Proc Natl Acad Sci U S A. 2013;110:324–329. - PMC - PubMed
1. Vesper O, et al. Selective Translation of Leaderless mRNAs by Specialized Ribosomes Generated by MazF in Escherichia coli. Cell. 2011;147:147–157. - PMC - PubMed
1. Xue S, Barna M. Specialized ribosomes: a new frontier in gene regulation and organismal biology. Nat Rev Mol Cell Biol. 2012;13:355–369. - PMC - PubMed
1. Dinman JD. The eukaryotic ribosome: current status and challenges. J Biol Chem. 2009;284:11761–5. - PMC - PubMed

Publication types

Actions
Actions

MeSH terms

Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions

Substances

Actions
Actions
Actions
Actions
Actions

Associated data

Actions
- Search in PubMed
- Search in Nucleotide

Grants and funding

LinkOut - more resources

Full Text Sources
Other Literature Sources
Molecular Biology Databases
- Mouse Genome Informatics (MGI)
- ZFIN
Research Materials
- Addgene Non-profit plasmid repository
- NCI CPTC Antibody Characterization Program
Miscellaneous
- NCI CPTAC Assay Portal

[1] Kondrashov N, et al. Ribosome-Mediated Specificity in Hox mRNA Translation and Vertebrate Tissue Patterning. Cell. 2011;145:383–397. - PMC - PubMed

[2] Kondrashov N, et al. Ribosome-Mediated Specificity in Hox mRNA Translation and Vertebrate Tissue Patterning. Cell. 2011;145:383–397. - PMC - PubMed

[3] Lee A, Burdeinick-Kerr R, Whelan S. A ribosome-specialized translation initiation pathway is required for cap-dependent translation of vesicular stomatitis virus mRNAs. Proc Natl Acad Sci U S A. 2013;110:324–329. - PMC - PubMed

[4] Lee A, Burdeinick-Kerr R, Whelan S. A ribosome-specialized translation initiation pathway is required for cap-dependent translation of vesicular stomatitis virus mRNAs. Proc Natl Acad Sci U S A. 2013;110:324–329. - PMC - PubMed

[5] Vesper O, et al. Selective Translation of Leaderless mRNAs by Specialized Ribosomes Generated by MazF in Escherichia coli. Cell. 2011;147:147–157. - PMC - PubMed

[6] Vesper O, et al. Selective Translation of Leaderless mRNAs by Specialized Ribosomes Generated by MazF in Escherichia coli. Cell. 2011;147:147–157. - PMC - PubMed

[7] Xue S, Barna M. Specialized ribosomes: a new frontier in gene regulation and organismal biology. Nat Rev Mol Cell Biol. 2012;13:355–369. - PMC - PubMed

[8] Xue S, Barna M. Specialized ribosomes: a new frontier in gene regulation and organismal biology. Nat Rev Mol Cell Biol. 2012;13:355–369. - PMC - PubMed

[9] Dinman JD. The eukaryotic ribosome: current status and challenges. J Biol Chem. 2009;284:11761–5. - PMC - PubMed

[10] Dinman JD. The eukaryotic ribosome: current status and challenges. J Biol Chem. 2009;284:11761–5. - PMC - PubMed

Save citation to file

Email citation

Add to Collections

Add to My Bibliography

Your saved search

Create a file for external citation management software

Your RSS Feed

RNA regulons in Hox 5' UTRs confer ribosome specificity to gene regulation

Affiliations

RNA regulons in Hox 5' UTRs confer ribosome specificity to gene regulation

Authors

Affiliations

Abstract

Conflict of interest statement

Figures

Comment in

Similar articles

Cited by

References

Publication types

MeSH terms

Substances

Associated data

Grants and funding

LinkOut - more resources

Full Text Sources

Other Literature Sources

Molecular Biology Databases

Research Materials

Miscellaneous

Abstract

Conflict of interest statement

Figures

Comment in

Similar articles

Cited by

References

Publication types

MeSH terms

Substances

Associated data

Related information

Grants and funding

LinkOut - more resources

Full Text Sources

Other Literature Sources

Molecular Biology Databases

Research Materials

Miscellaneous