Small changes in ambient temperature affect alternative splicing in Arabidopsis thaliana

Figures & data

Figure 1: Temperature dependence of AS events that change upon increasing and decreasing ambient temperature. (A) APUM23; (B) mTERF; (C) FYD; (D) AKIN11.The gene structure, the structure of the transcript isoforms around the AS event and the ratios of the two splice isoforms at each temperature are shown. On the left side of each panel, the percentage of each splice form +/− s.d. based on three biological replicates is indicated for each temperature. On the right side of each panel, the gene and transcript structures and the AS events are shown schematically. Exons are indicated by open boxes; UTRs, black rectangles; introns, thin lines; splicing events, diagonal lines. The arrowheads denote the approximate position of primers and the sizes of the PCR products from each splice isoform are indicated.

Table 1: Details of the AS events analyzed.

primer pair	AGI	Gene description	AS event	5′ primer	3′ primer
12	At1g72320	APUM23 (ARABIDOPSIS PUMILIO 23); RNA binding	3'SS	CGTCAACTGTGTTTTTGCATCC	CATCAAATCCACGGTTACCC
36	At4g12790	ATP-binding family protein	3'SS	GGTTTTGAGAGCAAAGAAAAACG
59	At5g66010	RNA-binding (RRM/RBD/RNP motifs) family protein	3'SS	GGCGGCAGGTCATGTACGG
72	At2g04790	similar to unnamed protein product [Vitis vinifera]	5'SS	CCCTGAAAGCATAGAAGCAGC	CCCATGACTTATTAAACTCC
75	At2g36000	mitochondrial transcription termination factor-related	5'SS	CTCGTTTAGTTTGGAGAATC	CTTCATCAGCATTCATTAC
87	At4g35450	AKR2 (ANKYRIN REPEAT-CONTAINING PROTEIN 2)	5'SS	CCACCACAACATTGTCTTTTC	CCAGCGTTAGGAATAGATCTC
118	At2g02960	zinc finger (C3HC4-type RING finger) family protein	3'SS	GGGGAGCTTTCACCAATTAG	GCCTTATCATTAACCACCGG
148	At1g76510	ARID/BRIGHT DNA-binding domain-containing protein	5'SS	CCGTTTCTCGCTTCTTTTTCTC	CCTCTACAACACCTTTGGTACC
179	At5g48150	PAT1 (PHYTOCHROME A SIGNAL TRANSDUCTION 1)	ES	CCTTGTCTCCGACAACTTTC
181	At5g05550	sequence-specific DNA binding transcription factors	ES	GGAGAAGCAGAGAATGGAAG	GGATCCTCCAATTTCAATGAG
187	At5g02470	DPA; transcription factor	5'SS	CAGTTTGTTTGTTTGTTTATAG	CCAATTTCAGAATCATCATC
189	At5g43270	SPL2 (SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 2)	5'SS	CCTCTGGGATCCATAAGTTTTG	CCATCAATTTCCCACTCCATTG
227	At4g24740	AFC2 (ARABIDOPSIS FUS3-COMPLEMENTING GENE 1)	ES	CCTCATACTCACATGGATCGTCGTCC
243	At2g33830	dormancy/auxin associated family protein	3'SS	CCGGACCTAAACCGGAGCATGGCC
261	At4g10100	CNX7/SIR5	5SS	CTCATGTGTGTGGTATTCACC	CAGTGTTAGATCAGGCACACC
268	At1g03457	RNA-binding (RRM/RBD/RNP motifs) family protein	3'SS	CCGTTGCAAGTTAAGTATGC	CCCTCTTAGAATCTGTAGATCC
285	At3g19840	pre-mRNA-processing protein 40C	5'SS	CCATATTCTGGTTCTCATCC	CCAGGCATCTAACCGATTTCC
288	At3g12570	FYD	3'SS	CCATGTGTTGTACTAGTGCC	CCATGGATAGCAGTGTTGAC
295	At2g02390	ATGSTZ1 (GLUTATHIONE S-TRANSFERASE 18)	3'SS	GGCTTGATTATGAGTATATACCAG
309	At5g65060	MAF3 (MADS AFFECTING FLOWERING 3)	3'SS	CAAGGAGTTACTAGAAATAGTCC	CCCGTGACATTCCTCTGTCACC
310	At5g65070	MAF4 (MADS AFFECTING FLOWERING 4)	5'SS	CCGTCGCTCTTATCATCATCTC
314	At2g43410	FPA	IR	GGGCTGGCTCTTACGATAACAG	GATGGCCTCCTCCAGTTTGG
322	At2g33480	ANAC041 (Arabidopsis NAC domain containing protein 41)	5'SS	CCGATGTTTGTAAATCCGATCC	CTGTCTCTTTCTCATTCTCC
324	At5g43270	SPL2 (SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 2)	5'SS	GGGATCCATAAGTTTTGAG	GCTTGAAGAATACAGAGAGG
327	At5g59950	RNA and export factor-binding protein, putative	IR	CTGCTCCATACCAATCAGCC	CCACTTCTATCAAAATGAAC
343	At3g29160	AKIN11 (ARABIDOPSIS SNF1 KINASE HOMOLOG 11)	5'SS	CCTGACTCAGCTCTGCGTCACC	CCCAATTCCAAGAGTTTTACC
378	At3g62190	DNAJ heat shock N-terminal domain-containing protein	3'SS	CCTGATGATCAGAAGCTTGTTGCC	CCGAGGAACCCCAGTCTTGAC
380	At5g08185	npcRNA 78; MIR162a	ES	GTCCATTTGGTTTCATAAGG

The number of the primer pair,^{6, 20} Arabidopsis gene identifier, gene description, AS event (3'SS, alternative 3’ splice site; 5′SS, alternative 5’ splice site; ES, exon skipping; IR, intron retention) and sequences of the primers are indicated.

Table 2: Transcripts with changes in AS patterns upon moderate temperature shifts.

A. Changes upon transfer of Col plants from 20°C to 16°C.							Total transcript/	Total transcript/	Fold change of
			Product sizes	Small product	Small product	P-value	RPL12C	RPL12C	Total transcript	P-value
Primer pair	AGI	Description	[BP]	[%] at 16 °C	[%] at 20 °C	20-16	Levels 16°C	Levels 20°C	Upon transfer to 16°C
12	At1g72320	APUM23	141 / 150 / 215	26	18	0.00175	0.0445	0.0608	0.73	0.40274
72	At2g04790	unknown protein	167 / 190	85	77	0.03573	0.5868	0.7230	0.81	0.36801
75	At2g36000	mitochondrial transcription termination factor-related	150 / 254	28	33	0.00014	0.1326	0.3745	0.35	0.16424
118	At2g02960	zinc finger (C3HC4-type RING finger) family protein	197 / 203 / 222	58	65	0.00737	0.0351	0.0424	0.83	0.39887
148	At1g76510	ARID/BRIGHT DNA-binding domain-containing protein	189 / 212	44	32	0.01842	0.0754	0.1275	0.59	0.00808
189	At5g43270	SPL2 SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 2	160 / 244	43	64	0.04011	0.0201	0.0271	0.74	0.83124
243	At2g33830	dormancy/auxin associated family protein	140 / 146	8	13	0.00902	1.8394	2.8719	0.64	0.07320
285	At3g19840	pre-mRNA-processing protein 40C	171 / 207	46	64	0.01337	0.0042	0.0070	0.60	0.47984
288	At3g12570	FYD	159 / 188	44	39	0.05332	0.0240	0.0428	0.56	0.02980
314	At2g43410	FPA	409 / 538	20	24	0.00774	0.0580	0.0851	0.68	0.08719
322	At2g33480	ANAC041 Arabidopsis NAC domain containing protein 41	321 / 399	10	15	0.00555	0.0579	0.0733	0.79	0.49148
343	At3g29160	AKIN11 ARABIDOPSIS SNF1 KINASE HOMOLOG 11	159 / 307	45	34	0.00804	0.1125	0.1437	0.78	0.24488
B Changes upon transfer of Col plants from 20°C to 24°C.							Total transcript/	Total transcript/	Fold change of
			product sizes	small product	small product	p-value	RPL12C	RPL12C	Total transcript	P-value
Primer pair	AGI	Description	[BP]	[%] at 20 °C	[%] at 24 °C	20-24	Levels 20°C	Levels 24°C	Upon transfer to 24°C
12	At1g72320	APUM23	141 / 150 / 215	18	14	0.04286	0.0608	0.0406	0.67	0.27551
36	At4g12790	ATP-binding family protein	212 / 338	41	46	0.03911	0.2433	0.2031	0.83	0.18972
59	At5g66010	RNA-binding (RRM/RBD/RNP motifs) family protein	105 /182	30	34	0.05018	0.0649	0.0719	1.11	0.62294
72	At2g04790	unknown protein	167 / 190	77	84	0.03198	0.7230	1.1533	1.60	0.00739
75	At2g36000	mitochondrial transcription termination factor-related	150 / 254	33	45	0.00003	0.3745	0.3893	1.04	0.89047
118	At2g02960	zinc finger (C3HC4-type RING finger) family protein	197 / 203 / 222	65	71	0.04727	0.0424	0.0419	0.99	0.95003

Changes in the ratio of AS isoforms with p <0.05 and changes >4 % are considered significant. In case more than two splice isoforms are generated the ones considered are underlined. The total transcript level of all alternative splice forms is expressed relative to the RPL12c transcript level. The fold-change between the levels observed at the different temperatures and the corresponding p-values are indicated.

Kalyna M, Simpson CG, Syed NH, Lewandowska D, Marquez Y, Kusenda B, et al. Alternative splicing and nonsense-mediated decay modulate expression of important regulatory genes in Arabidopsis. Nucleic Acids Res 2012; 40:2454 - 69; http://dx.doi.org/10.1093/nar/gkr932; PMID: 22127866

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