HeLa cells were transfected with plasmids expressing RNA-binding proteins and then cultured for 48 h, then treated with 10 ng/ml SSA for 3 h. Total lysate of the cells was analysed by western blotting to evaluate the phosphorylation status of Pol II. 
Pol IIo: hyperphosphorylated form; Pol IIa: hypophosphorylated form. (A, B) HeLa cells were transfected with a vector or chimeric plasmids, and then cultured for 48 h after transfection (A). HeLa cells were transfected with a vector or RNP deletion mutants, and then cultured for 48 h (B). The transfected cells were treated with SSA and 5-EU, and the labelled RNA was analysed as in Table S1 RNA binding proteins used in this study. The 23 RBPs which were used in Figure 1 were in red letters. 
SMEK2 Ex4-5 CDK6 Ex2-3 CDK6 Ex2 CDK6 Ex8 VEGFA Ex1 VEGFA Ex8 CDK6 Ex8/Ex2 VEGFA Ex8/Ex1

A1




  Fig. S1 Muraoka et al. 


Figure S1 .Figure S2 .

 S1S2 Figure S1. Protein expression of RNA-binding proteins.HeLa cells were transfected with plasmids expressing RNA-binding proteins and then cultured for 48 h. Total lysate of the cells was analysed by western blotting to evaluate the expression levels of the RNA binding proteins. 




  Fig. S3 Muraoka et al. 


Figure S3 .

 S3 Figure S3. The DNA sequence of the part of the SMEK2 intron 4 used for the RNA binding assay. The red letters indicate putative Rbm38 binding sequences. 


Figure S4 .

 S4 Figure S4. The effects of Rbm38 and Rbm24 on transcription elongation and splicing. (A, B, C) HeLa cells were transfected with a plasmid, Flag-Rbm24 or Flag-Rbm38, and then cultured for 48 h. The transfected cells were treated with SSA and 5-EU, and the labelled RNA was analysed as in Fig. 1. Error bars indicate S.D. (n = 3). Statistical significance was investigated by one-way ANOVA and Tukey' s test (*: p<0.05; **: p<0.01; ***: p<0.001). 


Figure S6 .

 S6 Figure S6. Exogenous Rbm38 WT and mutants are phosphorylated and truncated.(A, B) HeLa cells were transfected with Rbm38 plasmids and cultured for 48 h, Total lysate were prepared and treated with protein phosphatase. The lysates were analysed by western blotting. 


Fig. 1 .

 1 Figure S7. The N-and C-terminal regions and RBD/RRM domain of Rbm38 are important for suppressing the transcription elongation defect.(A, B) HeLa cells were transfected with a vector or chimeric plasmids, and then cultured for 48 h after transfection (A). HeLa cells were transfected with a vector or RNP deletion mutants, and then cultured for 48 h (B). The transfected cells were treated with SSA and 5-EU, and the labelled RNA was analysed as in Fig. 1. Error bars indicate S.D. (n = 3). Statistical significance was investigated by one-way ANOVA and Tukey' s test (*: p<0.05; **: p<0.01; ***: p<0.001). 


Figure. S8 .

 S8 Figure. S8. Images of the full-length, unprocessed membranes and western blots shown in Fig. 2C. 


Figure. S12 .

 S12 Figure. S12. Images of the full-length, unprocessed western blots shown in Fig. S6. 




  




  




  




  




  




  


Table S2 Top10

 S2 Rbm38 binding motifs reported byHeinicke et al. (Ref. 22) 


	GTGTGTG
	TGTGTGT
	GTGTGTT
	GGTGTGT
	AGTGTGT
	CGTGTGT
	TTGTGTG
	GTTGTGT
	GTGTGGT
	TGGTGTG

Table S3 .

 S3 List of primers used in this study. 


	For cloning	
	Name	Sequence
	mRBM38F	CCCGAATTCATGCTGCTGCAGCCCG
	mRBM38R	CCCCTCGAGCTGCATCCTGTCAGGCTGTAG
	RBM38∆NF	CCCGAATTCATGACCAAGATCTTCGTGGGCG
	RBM38∆CR	CCCCTCGAGCAGGTAGGCCAGGTTCACATTG
	Chimera1F	CGAATTCATGCACACCACCCAGAAGGACACGACGTAC ACCAAGATCTTCGTGGGCGGCCT
	Chimera2R	TGCTCCCAAGTATGCCAGGTTCACATTGGCCTTGCG
	Chimera3F	CGCAAGGCCAATGTGAACCTGGCATACTTGGGAGCA
	mRBM24R	CCCCTCGAGCTGCATTCGGTCTGTCTGC
	∆RNP1F	CGGCAAGTCCGCAGATCGGGCAGCGGC
	∆RNP1R	CCCGATCTGCGGACTTGCCGGTCTGGC
	∆RNP2F	GTTCACCAAGCCCTACCACACCACCGAC
	∆RNP2R	TGTGGTAGGGCTTGGTGAACGTGGTGTC
	SMEK2 int4 cloning for EcoRI	GCCGCCGAATTCAAATGTGTGTGTAGGCTGGGTG
	SMEK2 int4 cloning rev XhoI-3	GCCGCCCTCGAGGCACCCAGTTCAGTACCTTAGC
	For RT-qPCR	
	Name	Sequence
	18S rRNA for	GTTGGTGGAGCGATTTGTCTGGTT
	18S rRNA rev	TATTGCTCAATCTCGGGTGGCTGA
	SMEK2 Ex3 for	TGCCTTTACCGTCTCCTAAGAGTG
	SMEK2 Ex3 rev	ATTGCCGGTCTTCGTTCAGGGTAT
	SMEK2 Ex5 for	TGTTTGGTCAGAAGCAGAGAA
	SMEK2 Ex5 rev	TTTCCCAGATCTCATCACAGC
	SMEK2 Ex19 for	GGAAGTTTGGTTGGCTTAGTGG
	SMEK2 Ex19 rev	GCTCACTGAACAGTTGCAGCATTG
	CDK6 Ex2 for	AGTACGAATGCGTGGCGGAGAT
	CDK6 Ex2 rev	AAACGGCCTCCGTTCTTCAAGT
	CDK6 Ex8 for	CTGCTGACCAATTGTGCTGCCATT
	CDK6 Ex8 rev	CACACACACATGCACACACACACT
	VEGF Ex1 for	GAGGCGCAGCGGTTAGGT
	VEGF Ex1 rev	CGGATCAATGAATATCAAATTCCA
	VEGF Ex8 for	CTGGCGCTGAGCCTCTCTAC
	VEGF Ex8 rev	CCGGTGTCCTCATCCCTGTA
	CDK6 Ex2-3 spliced for	AGGCACCTGGAGACCTT
	CDK6 Ex2-3 spliced rev	TGGTTTCTCTGTCTGTTCGTG
	SMEK2 Ex4-5 unspliced for	CCCTATTCTACAGTTCCGATAACC
	SMEK2 Ex4-5 unspliced rev	AGTTCTCTGCTTCTGACCAAAC

Supplementary Information

Rbm38 reduces transcription elongation defect of SMEK2 gene caused by splicing deficiency
Shintaro Muraoka, Kazuhiro Fukumura, Megumi Hayashi, Naoyuki Kataoka, Akila Mayeda, Daisuke Kaida