significantly different, mix them at a ratio that equalizes their
signal strength. Use a 1:100 dilution of the marker mix as your
working stock. At each step estimate an appropriate marker
that needs to be used to fit the signal in your samples. Marker
signal decays with time at
32
P half-life rate (t 1/2 ~ 2 weeks).
References
1. Wilk R, Hu J, Blotsky D, Krause HM (2016)
Diverse and pervasive subcellular distributions
for both coding and long noncoding RNAs.
Genes Dev 30:594–609. https://doi.org/10.
1101/gad.276931.115
2. Kejiou NS, Palazzo AF (2017) mRNA localization as a rheostat to regulate subcellular gene
expression. Wiley Interdiscip Rev RNA:e1416.
https://doi.org/10.1002/wrna.1416
3. Le ´cuyer E, Yoshida H, Parthasarathy N et al
(2007) Global analysis of mRNA localization
reveals a prominent role in organizing cellular
architecture and function. Cell 131:174–187.
https://doi.org/10.1016/j.cell.2007.08.003
4. Hung V, Udeshi ND, Lam SS et al (2016)
Spatially resolved proteomic mapping in living
cells with the engineered peroxidase APEX2.
Nat Protoc 11:456–475. https://doi.org/10.
1038/nprot.2016.018
5. Li Y, Aggarwal MB, Ke K et al (2018)
Improved analysis of RNA localization by spatially restricted oxidation of RNA-protein complexes. Biochemistry 57:1577–1581. https://
doi.org/10.1021/acs.biochem.8b00053
6. Ferna ´ndez-Sua ´rez M, Chen TS, Ting AY
(2008) Protein-protein interaction detection
in vitro and in cells by proximity biotinylation.
J Am Chem Soc 130:9251–9253. https://doi.
org/10.1021/ja801445p
7. Yang J, Jaramillo A, Shi R et al (2004) In vivo
biotinylation of the major histocompatibility
complex (MHC) class II/peptide complex by
coexpression of BirA enzyme for the generation of MHC class II/tetramers. Hum Immunol 65:692–699. https://doi.org/10.1016/j.
humimm.2004.04.001
8. de Boer E, Rodriguez P, Bonte E et al (2003)
Efficient biotinylation and single-step purification of tagged transcription factors in mammalian cells and transgenic mice. Proc Natl Acad
Sci U S A 100:7480–7485. https://doi.org/
10.1073/pnas.1332608100
9. O’callaghan CA, Byford MF, Wyer JR et al
(1999) BirA enzyme: production and application in the study of membrane receptor-ligand
interactions by site-specific biotinylation. Anal
Biochem 266:9–15. https://doi.org/10.
1006/abio.1998.2930
10. Fazal FM, Han S, Kaewsapsak P, et al (2018)
Atlas of subcellular RNA localization revealed
by APEX-seq. bioRxiv 454470. https://doi.
org/10.1101/454470
11. Kaewsapsak P, Shechner DM, Mallard W et al
(2017) Live-cell mapping of organelleassociated RNAs via proximity biotinylation
combined with protein-RNA crosslinking. elife
6:623. https://doi.org/10.7554/eLife.29224
12. Padron A, Iwasaki S, Ingolia N (2018) Proximity RNA labeling by APEX-Seq reveals the
organization of translation initiation complexes
and repressive RNA granules. bioRxiv 454066.
https://doi.org/10.1101/454066
13. Benhalevy D, Anastasakis DG, Hafner M
(2018) Proximity-CLIP provides a snapshot
of protein-occupied RNA elements in subcellular
compartments.
Nat
Methods
15:1074–1082.
https://doi.org/10.1038/
s41592-018-0220-y
14. Benhalevy D, Anastasakis D, Hafner M (2018)
Proximity-CLIP. Protoc Exch. https://doi.
org/10.1038/protex.2018.115
15. Koch L (2019) Proximity-CLIP - close
encounters of the RNA kind. Nat Rev Genet
20:68–69. https://doi.org/10.1038/s41576018-0086-y
16. Choder M (2011) mRNA imprinting: additional level in the regulation of gene expression. Cell Logist 1:37–40. https://doi.org/
10.4161/cl.1.1.14465
17. Hafner M, Landthaler M, Burger L et al (2010)
Transcriptome-wide
identification
of
RNA-binding protein and microRNA target
sites by PAR-CLIP. Cell 141:129–141.
https://doi.org/10.1016/j.cell.2010.03.009
18. Benhalevy D, McFarland HL, Sarshad AA,
Hafner M (2016) PAR-CLIP and streamlined
small RNA cDNA library preparation protocol
for the identification of RNA binding protein
target sites. Methods 118-119:41–49. https://
doi.org/10.1016/j.ymeth.2016.11.009
19. Branon TC, Bosch JA, Sanchez AD et al
(2018) Efficient proximity labeling in living
cells and organisms with TurboID. Nat Biotechnol 36:880–887. https://doi.org/10.
1038/nbt.4201
Proximity-CLIP
305
signal strength. Use a 1:100 dilution of the marker mix as your
working stock. At each step estimate an appropriate marker
that needs to be used to fit the signal in your samples. Marker
signal decays with time at
32
P half-life rate (t 1/2 ~ 2 weeks).
References
1. Wilk R, Hu J, Blotsky D, Krause HM (2016)
Diverse and pervasive subcellular distributions
for both coding and long noncoding RNAs.
Genes Dev 30:594–609. https://doi.org/10.
1101/gad.276931.115
2. Kejiou NS, Palazzo AF (2017) mRNA localization as a rheostat to regulate subcellular gene
expression. Wiley Interdiscip Rev RNA:e1416.
https://doi.org/10.1002/wrna.1416
3. Le ´cuyer E, Yoshida H, Parthasarathy N et al
(2007) Global analysis of mRNA localization
reveals a prominent role in organizing cellular
architecture and function. Cell 131:174–187.
https://doi.org/10.1016/j.cell.2007.08.003
4. Hung V, Udeshi ND, Lam SS et al (2016)
Spatially resolved proteomic mapping in living
cells with the engineered peroxidase APEX2.
Nat Protoc 11:456–475. https://doi.org/10.
1038/nprot.2016.018
5. Li Y, Aggarwal MB, Ke K et al (2018)
Improved analysis of RNA localization by spatially restricted oxidation of RNA-protein complexes. Biochemistry 57:1577–1581. https://
doi.org/10.1021/acs.biochem.8b00053
6. Ferna ´ndez-Sua ´rez M, Chen TS, Ting AY
(2008) Protein-protein interaction detection
in vitro and in cells by proximity biotinylation.
J Am Chem Soc 130:9251–9253. https://doi.
org/10.1021/ja801445p
7. Yang J, Jaramillo A, Shi R et al (2004) In vivo
biotinylation of the major histocompatibility
complex (MHC) class II/peptide complex by
coexpression of BirA enzyme for the generation of MHC class II/tetramers. Hum Immunol 65:692–699. https://doi.org/10.1016/j.
humimm.2004.04.001
8. de Boer E, Rodriguez P, Bonte E et al (2003)
Efficient biotinylation and single-step purification of tagged transcription factors in mammalian cells and transgenic mice. Proc Natl Acad
Sci U S A 100:7480–7485. https://doi.org/
10.1073/pnas.1332608100
9. O’callaghan CA, Byford MF, Wyer JR et al
(1999) BirA enzyme: production and application in the study of membrane receptor-ligand
interactions by site-specific biotinylation. Anal
Biochem 266:9–15. https://doi.org/10.
1006/abio.1998.2930
10. Fazal FM, Han S, Kaewsapsak P, et al (2018)
Atlas of subcellular RNA localization revealed
by APEX-seq. bioRxiv 454470. https://doi.
org/10.1101/454470
11. Kaewsapsak P, Shechner DM, Mallard W et al
(2017) Live-cell mapping of organelleassociated RNAs via proximity biotinylation
combined with protein-RNA crosslinking. elife
6:623. https://doi.org/10.7554/eLife.29224
12. Padron A, Iwasaki S, Ingolia N (2018) Proximity RNA labeling by APEX-Seq reveals the
organization of translation initiation complexes
and repressive RNA granules. bioRxiv 454066.
https://doi.org/10.1101/454066
13. Benhalevy D, Anastasakis DG, Hafner M
(2018) Proximity-CLIP provides a snapshot
of protein-occupied RNA elements in subcellular
compartments.
Nat
Methods
15:1074–1082.
https://doi.org/10.1038/
s41592-018-0220-y
14. Benhalevy D, Anastasakis D, Hafner M (2018)
Proximity-CLIP. Protoc Exch. https://doi.
org/10.1038/protex.2018.115
15. Koch L (2019) Proximity-CLIP - close
encounters of the RNA kind. Nat Rev Genet
20:68–69. https://doi.org/10.1038/s41576018-0086-y
16. Choder M (2011) mRNA imprinting: additional level in the regulation of gene expression. Cell Logist 1:37–40. https://doi.org/
10.4161/cl.1.1.14465
17. Hafner M, Landthaler M, Burger L et al (2010)
Transcriptome-wide
identification
of
RNA-binding protein and microRNA target
sites by PAR-CLIP. Cell 141:129–141.
https://doi.org/10.1016/j.cell.2010.03.009
18. Benhalevy D, McFarland HL, Sarshad AA,
Hafner M (2016) PAR-CLIP and streamlined
small RNA cDNA library preparation protocol
for the identification of RNA binding protein
target sites. Methods 118-119:41–49. https://
doi.org/10.1016/j.ymeth.2016.11.009
19. Branon TC, Bosch JA, Sanchez AD et al
(2018) Efficient proximity labeling in living
cells and organisms with TurboID. Nat Biotechnol 36:880–887. https://doi.org/10.
1038/nbt.4201
Proximity-CLIP
305