Cloning and Mutagenesis of OTUD5 Variants

pDEST-FLAG-HA-USP5 and pDEST-FLAG-HA-OTUD5 were gifts from W. Harper (Addgene plasmids #22590 and #22610) (75 (link)). pRK5-HA-Ub-K48only and pRK5-HA-Ub-K63only were gifts from T. Dawson (Addgene plasmids #17605 and #1706). OTUD5 patient mutations (G494S, L352P, 161-164del, R274W, and D256N), point mutations (C224S or C224R), truncation mutations (ΔCterm = OTUD5 1-534, Cterm = OTUD5 534-571), and wobble mutations to make constructs resistant to shOTUD5#5 were introduced in this vector using the Q5 site-directed mutagenesis kit (E0554, NEB) following the manufacturer’s instructions. OTUD5 and OTUD5C224R were further subcloned into pKmyc using Bam HI and Not I sites (pKmyc-OTUD5 WT or C224R). For expression in hESCs, OTUD5 variants were cloned into pENTR1A or pENTR233 and recombined into pINDUCER20 (76 (link)). pLKO1-Puro Mission shRNA constructs targeting OTUD5 (#2: TRCN0000122275 and #5: TRCN0000233196), ARID1A (TRCN0000059092), ARID1B (TRCN00000420576), UBR5 (TRCN0000003411), HDAC2 (TRCN00000004819), HCFC1 (TRCN00000001625), TRAF3 (TRC00000034219), and TRIM25 (TRC0000003499) were purchased from Sigma-Aldrich. siRNA pools were purchased from Santa Cruz Biotechnology and Thermo Fisher Scientific.

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Beck D.B., Basar M.A., Asmar A.J., Thompson J.J., Oda H., Uehara D.T., Saida K., Pajusalu S., Talvik I., D’Souza P., Bodurtha J., Mu W., Barañano K.W., Miyake N., Wang R., Kempers M., Tamada T., Nishimura Y., Okada S., Kosho T., Dale R., Mitra A., Macnamara E., Matsumoto N., Inazawa J., Walkiewicz M., Õunap K., Tifft C.J., Aksentijevich I., Kastner D.L., Rocha P.P, & Werner A. (2021). Linkage-specific deubiquitylation by OTUD5 defines an embryonic pathway intolerant to genomic variation. Science Advances, 7(4), eabe2116.

Publication 2021

Q5 site-directed mutagenesis kit ARID1A HDAC2 HCFC1 siRNA pools

Arid1a Gifts Hescs Mutations Otud5 Patient Plasmids Point mutations Shrna Sirna Site directed mutagenesis Traf3 Trim25 Vector

Corresponding Organization :

Other organizations : National Human Genome Research Institute, National Institutes of Health, National Institute of Dental and Craniofacial Research, Tokyo Medical and Dental University, Yokohama City University, Tartu University Hospital, University of Tartu, Yale University, Tallinn Health Care College, Johns Hopkins Hospital, Children's Hospital of Orange County, University of California, Irvine, Radboud University Nijmegen, Radboud University Medical Center, Hiroshima Prefectural Rehabilitation Center, Hiroshima City Asa Citizens Hospital, Hiroshima University, Shinshu University, Office of the Director, National Institute of Allergy and Infectious Diseases, National Cancer Institute

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Variable analysis

independent variables

OTUD5 patient mutations (G494S, L352P, 161-164del, R274W, and D256N)
OTUD5 point mutations (C224S or C224R)
OTUD5 truncation mutations (ΔCterm = OTUD5 1-534, Cterm = OTUD5 534-571)
Wobble mutations to make constructs resistant to shOTUD5#5

dependent variables

Not explicitly mentioned

control variables

PDEST-FLAG-HA-USP5 and pDEST-FLAG-HA-OTUD5 plasmids from W. Harper (Addgene)
PRK5-HA-Ub-K48only and pRK5-HA-Ub-K63only plasmids from T. Dawson (Addgene)
PLKO1-Puro Mission shRNA constructs targeting OTUD5, ARID1A, ARID1B, UBR5, HDAC2, HCFC1, TRAF3, and TRIM25 from Sigma-Aldrich
SiRNA pools from Santa Cruz Biotechnology and Thermo Fisher Scientific

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

Annotations

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