REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
TTDN1 (Rabbit)	Novus	CAT#NBP2-31718
DBR1 (Rabbit)	Invitrogen	CAT#PA5-57244; RRID:AB_2640394
GAPDH (Rabbit)	Abcam	CAT#ab9485; RRID:AB_307275
Flag (Rabbit)	Sigma	CAT#F7425; RRID:AB_439687
HA (Mouse)	BioLegend	CAT#901501; RRID:AB_2565006
AQR (Rabbit)	Bethyl Labs	CAT#A302-546A; RRID:AB_1998969
XAB2 (Rabbit)	Bethyl Labs	CAT#A303-638A; RRID:AB_11205112
ZNF830 (Rabbit)	Bethyl Labs	CAT#A301-419A; RRID:AB_960966
PRP8 (Rabbit)	Bethyl	CAT#A303-922A; RRID:AB_2620271
PRP19 (Rabbit)	Sigma	CAT#SAB4501215; RRID:AB_10745273
PRP43 (Rabbit)	Abcam	CAT#ab254591 RRID:AB_2892059
SF3A1 (Rabbit)	Bethyl Labs	CAT#A301-601A; RRID:AB_1078817
IgG (Rabbit)	Santa Cruz	CAT#sc-2027; RRID:AB_737197
Chemicals, peptides, recombinant proteins
GST	(Soll et al., 2018)	N/A
GST-TTDN1	This study	N/A
GST-TTDN1-Aro>Ala	This study	N/A
GST-ASCC1	(Soll et al., 2018)	N/A
Flag-MBP-TTDN1-mCherry	This study	N/A
Flag-MBP-TTDN1-Aro>Ala-mCherry	This study	N/A
mCherry	Biovision	4993
His-prp-DBR1-GFP	This study	N/A
His-Flag-DBR1	This study	N/A
His-AQR	This study	N/A
Recombinant intron binding complex (IBC)	This study	N/A
Murine Rnase Inhibitor	NEB	M0314L
Rnase-free Dnase	Qiagen	79256
Flag peptide	Sigma	F3290
Anti-Flag M2 agarose beads	Sigma	A2220
Anti-HA agarose beads	Santa Cruz	sc-7392 AC
Anti-mCherry Nanobody Affinity Gel	Biolegend	689502
Pladienolide-B	Cayman Chemical Company	16538
Puromycin	Sigma	P8833
Blasticidin S	Sigma	15205
Protease & phosphatase inhibitor cocktail	Thermo Fisher Scientific	A32961
Ni-NTA beads	Qiagen	88221
Hoechst 33342	BD Bioscience	561908
ProLong Gold	Invitrogen	P36930
Critical Commercial Assays
miRNeasy mini kit	Qiagen	217004
High capacity cDNA synthesis kit	Thermo Fisher Scientific	4368814
SYBR Green JumpStart Taq Ready Mix	Sigma	S9194
SuperScript VILO cDNA synthesis kit	Thermo Fisher Scientific	11754050
DreamTaq Green PCR Master Mix	Thermo Fisher Scientific	KK1081
Deposited Data
RNA sequencing data	This paper	GEO (Accession: GSE233383)
Proteomics data	This paper	PRIDE (Accession: PXD042563, PXD042580, PXD042581)
Source images and original blots	This paper	Mendeley (DOI: 10.17632/prgb7kwjz8.1)
Experimental models: Cell lines
293T	ATCC	N/A
HeLa-S	ATCC	N/A
U2OS	ATCC	N/A
U2OS TTDN1 KO	This study	N/A
HeLa-S TTDN1 KO	This study	N/A
U2OS WT/WT	This study	N/A
U2OS Ala/KO	This study	N/A
HeLa-S DBR1 KO	This study	N/A
Fibroblast: Male TTDN1 2BP DEL, 187GG	Coriell Institute for Medical Research	GM06331
Fibroblast: Female TTDN1 2BP DEL, 187GG	Coriell Institute for Medical Research	GM06332
Fibroblast: Female TTDN1 2BP DEL, 187GG	Coriell Institute for Medical Research	GM06333
Fibroblast: Female unaffected	Coriell Institute for Medical Research	GM016648
Fibroblast: male unaffected	Coriell Institute for Medical Research	GM016650
Experimental models: Mouse
Ttdn1 Δ/Δ	This Study	N/A
Ttdn1 M143V/M143V	This Study	N/A
Oligonucleotides
POU6F2 (forward): 5’-CAAGCATCCATGTCTCAAAGTC-3’	IDT	N/A
POU6F2 (reverse): 5’-ATTAACCCCATCCACCTCAC-3’	IDT	N/A
TLL1 (forward): 5’-AAAGAAGTGATGAAGAGAGTTACATTG-3’	IDT	N/A
TLL1 (reverse): 5’-GCCGATAGAGATTGCCTGAG-3’	IDT	N/A
BCR (forward): 5’-AATGGCTGAGAAGTGCTGT-3’	IDT	N/A
BCR (reverse): 5’-CTTGTAGAGCAGAGTTTCCAGAG-3’	IDT	N/A
DHRS2 (forward): 5’-TGAGACCATCACCAAGCG-3’	IDT	N/A
DHRS2 (reverse): 5’-TCACAGAAAGCCTAGCACAG-3’	IDT	N/A
MLPH (forward): 5’-CGGAAGTGCCAAGGTCATC-3’	IDT	N/A
MLPH (reverse): 5’-GTTCTCCATCCTCATCTGTCTG-3’	IDT	N/A
SRGN (forward): 5’-GAAGCTACTCAAATGCAGTCG-3’	IDT	N/A
SRGN (reverse): 5’-ACCCATTGGTACCTGGCT-3’	IDT	N/A
STMN2 (forward): 5’-CCACGAACTTTAGCTTCTCCA-3’	IDT	N/A
STMN2 (reverse): 5’-GCCAATTGTTTCAGCACCTG-3’	IDT	N/A
β-actin (forward): 5’-CCAACCGCGAGAAGATGA-3’	IDT	N/A
β-actin (reverse): 5’-CCAGAGGCGTACAGGGATAG-3’	IDT	N/A
UBA1–1st run (forward): 5’-GGCTTCCCCACTTCCAG-3’	IDT	N/A
UBA1–1st run (reverse): 5’-GAAGTGAGGGAGGGAGG-3’	IDT	N/A
UBA1–2nd run (forward): 5’- CTGTGCGCCTTGTACTT-3’	IDT	N/A
UBA1–2nd run (reverse): 5’-TCTTACACTTAGGCATGCAAC-3’	IDT	N/A
RPL29–1st run (forward): 5’- GGTATTTCCACATTTGAGGTTG-3’	IDT	N/A
RPL29–1st run (reverse): 5’-AGGAATTGCAGGCTTTGG-3’	IDT	N/A
RPL29–2nd run (forward): 5’-ATGCTGTCTTTAGAATGCTGG-3’	IDT	N/A
RPL29–2nd run (reverse): 5’-GCCAGTTAGGCTGTGC-3’	IDT	N/A
Recombinant DNA
pET-28a-Flag-DBR1	This study	N/A
pFastBac-GST-TTDN1	This study	N/A
pGEX-ASCC1	(Soll et al., 2018)	N/A
pGEX-TTDN1	This study	N/A
pGEX-TTDN1-Aro>Ala	This study	N/A
MacroBac438-Flag-MBP-TTDN1-mCherry	This study	N/A
MacroBac438-Flag-MBP-TTDN1-Aro>Ala-mCherry	This study	N/A
His-prp-DBR1-GFP	This study	N/A
His-AQR	This study	N/A
pHAGE-3xHA-GFP	This study	N/A
pHAGE-3xHA-TTDN1	This study	N/A
pHAGE-Flag-TTDN1	This study	N/A
pHAGE-Flag-TTDN1 NΔ20	This study	N/A
pHAGE-Flag-TTDN1 NΔ60	This study	N/A
pHAGE-Flag-TTDN1 CR1–2	This study	N/A
pHAGE-Flag-TTDN1 ΔCR1–2	This study	N/A
pHAGE-Flag-TTDN1 ΔCR2	This study	N/A
pHAGE-Flag-TTDN1 E135K, E137K	This study	N/A
pHAGE-Flag-TTDN1 K141E	This study	N/A
pHAGE-Flag-TTDN1 E146K, D147K	This study	N/A
pHAGE-Flag-TTDN1 E153K	This study	N/A
pMSCV-Flag-HA-DBR1	This study	N/A
pMSCV-Flag-HA-TTDN1	This study	N/A
pHAGE-Flag-TTDN1 Aro>Ala	This study	N/A
pHAGE-Flag-DBR1-TTDN1 IDR	This study	N/A
pHAGE-Flag-AQR	This study	N/A
pLentiCRISPR-V2 -TTDN1	This study	N/A
pLentiCRISPR-V2 -DBR1	This study	N/A
pLentiCRISPR-V2 -NTC	Tsao et al., 2021	N/A
Software and algorithms
ImageLab	BioRad	N/A
ImageJ	https://imagej.nih.gov	N/A
rMATS turbo v4.1.1	https://zenodo.org/badge/latestdoi/644252407	N/A
Gene length	https://zenodo.org/record/7947667	N/A
Branchpoint detection algorithm	Pineda et al., 2018	N/A
Photoshop	Adobe	N/A
GraphPad Prism	N/A	N/A
Stereo Investigator Software	Version 2020.2.3, MBF Bioscience, Williston, VT	N/A
Sequest software	Eng et al., 1994	N/A