Splicing quality control mediated by DHX15 and its G-patch activator SUGP1

. Author manuscript; available in PMC: 2024 Feb 5.

Published in final edited form as: Cell Rep. 2023 Oct 8;42(10):113223. doi: 10.1016/j.celrep.2023.113223

KEY RESOURCES TABLE

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies

Anti-HA	MilliporeSigma	Cat#: 3F10; RRID:AB_2314622
Anti-FLAG (M2)	MilliporeSigma	Cat#: F3165; RRID:AB_259529
Anti-DHX15	Abcam	Cat#: Ab254591; RRID:AB_2892059
Anti-β-actin (AC-15)	MilliporeSigma	Cat#: A5441; RRID:AB_476744
Anti-H3	Abcam	Cat#: Ab1791; RRID:AB_302613
Anti-SC35	Abcam	Cat#: Ab204916; RRID:AB_2909393
Anti-SF3B1	Abcam	Cat#: Ab170854
Anti-U2AF2	Invitrogen	Cat#: PA530442; RRID:AB_2547916
Anti-U2AF1	Proteintech	Cat#: 10334-1 AP; RRID:AB_2211314
IRdye 800CW Sterptavidin	LI-COR	Cat#: 926-32230
Anti-mouse Alexa Fluor 488	ThermoFisher	Cat#: A28175; RRID:AB_2536161
Anti-rat Alexa Fluor 647	ThermoFisher	Cat#: A21247; RRID:AB_141778
NeutrAvidin- DyLight 594	ThermoFisher	Cat#: 22842

Chemicals, peptides, and recombinant proteins

dTAG13	Tocris Bioscience	Fisher Scientific Cat#: 66-055
α-amanitin	Santa Cruz Biotechnology	Cat#: Sc-202440A
cOmplete^™ ULTRA protease inhibitor	Roche	MilliporeSigma Cat#: 589297001
PhosStop	Roche	MilliporeSigma Cat#: 4906837001
Hygromycin	Millipore	Cat#: 400052
Lipofectamine 3000	Invitrogen	Cat#: L3000008
Proteinase K	Viagen	Cat#: 501-PK
Direct PCR reagent	Viagen	Cat#: 301-C
Direct-zol	Zymo Research	Cat#: R2052
SUPERaseIn	Life Technologies	Cat#: AM2694
Tri reagent	Zymo Research	Cat#: R2050-1-200
Doxycyclin	MilliporeSigma	Cat#: D9891
Biotin Phenol	MilliporeSigma	Cat#: SML2135-250MG
H2O2	MilliporeSigma	Cat#: H1009-100ML
Streptavidin-coated magnetic beads	Pierce	Thermo Scientific Cat#: 88817
DAPI	ThermoFisher	Cat#: 62248

Critical commercial assays

NEBuilder HiFi DNA assembly cloning kit	NEB	Cat#: E5520S
NEB OneTaq Quick-Load 2X Mastermix	NEB	Cat#: M0486L
NEBNext Poly(A) mRNA magnetic isolation module	NEB	Cat#: E7490L
NEBNext Ultra II Directional RNA kit	NEB	Cat#: E7760L
NEBNext rRNA Depletion Kit	NEB	Cat#: E6310X

Deposited data

Human reference genome NCBI release 28, GRCh38	Genome Reference Consortium	http://www.ncbi.nlm.nih.gov/projects/genome/assembly/grc/human/
SUGP1 KD	Liu et al.¹⁹	GEO: GSE159304
RBM10 KD	Wang et al.⁶²	GEO: GSE44976
SUGP2 KD	ENCODE Consortium	ENCLB206KMT, ENCLB331AGB
NKRF KD	ENCODE Consortium	ENCLB552FZS, ENCLB950ZAG
TFIP11 KD	ENCODE Consortium	ENCLB144PBT, ENCLB483ITG
RBM17 KD	ENCODE Consortium	ENCLB012PNW, ENCLB719FFS
GPKOW KD	ENCODE Consortium	ENCLB168TMK, ENCLB908ZJN
RBM5 (CRISPR KO)	ENCODE Consortium	ENCLB036OZM, ENCLB293XAV
AGGF (CRISPR KO)	ENCODE Consortium	ENCLB710LCN, ENCLB644OBV
Raw and analyzed data	This Paper	GEO: GSE241712

Experimental models: Cell lines

HEK293T.A2	Khandelia et al.⁶³	N/A
HEK.FLAG-AP-DHX15.WT	This paper	N/A
HEK.dDHX15	This paper	N/A
HEK.dDHX38	This paper	N/A

Oligonucleotides

See Table S1	N/A	N/A

Recombinant DNA

pSpCas9-gRNA-GFP	Ran et al.²⁷	Cat# PX458; no. 48138
pUC19-DHX15- FKBP^F36V-2xHA_P2A_BFP	This paper	N/A
pUC19-DHX15- FKBP^F36V-2xHA_P2A_HygR	This paper	N/A
pUC19-DHX38- FKBP^F36V-2xHA_P2A_BFP	This paper	N/A
pRD-FLAG-AP-DHX15 (wt and mutants)	This paper	N/A
pX304-HA-SUGP1-EX (full-length and truncations)	This paper	N/A
pCAGGS-nlCre	Khadelia et al.⁶³	N/A

Software and algorithms

STAR version 2.7.3.a	Dobin et al.⁶⁴	https://github.com/alexdobin/STAR
RSEM v1.3.3	Li et al.⁶⁵	https://github.com/deweylab/RSEM
Samtools V5.2.5	Li et al.⁶⁶	http://www.htslib.org/
clusterProfiler v4.0	Yu et al.⁶⁷	https://bioconductor.org/packages/release/bioc/html/clusterProfiler.html
MaxEntScan	Yeo et al.⁶⁸	http://hollywood.mit.edu/burgelab/maxent/Xmaxentscan_scoreseq.html
ComplexHeatmap v.2.2.0	Gu et al.⁶⁹	https://bioconductor.org/packages/release/bioc/html/ComplexHeatmap.html
Corrplot v0.92		https://github.com/taiyun/corrplot
ColabFold	Mirdita et al.⁴³	https://github.com/sokrypton/ColabFold
Pymol v2.5.2		https://pymol.org
Nikon NIS-Elements Imaging Software	Nikon	N/A
FIJI	Schindelin et al.⁷⁰	https://imagej.net/software/fiji/