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Supplementary Information
Specific splicing defects in S. pombe carrying a degron allele of the Survival of Motor
Neuron gene
Yannick Campion, Henry Neel, Thierry Gostan, Johann Soret and Remy Bordonné
Supplementary Figure 1
Characterization of the tdSMN allele
(A) Schematic representation of the tdSMN allele compared to the wild type SMN gene. The
grey boxes in wild type represent the regions used for homologous recombination. KANA:
G418 marker; nmt41: promoter; DHFR: dihydrofolate reductase; HA: anti-HA epitope;
SpSMN: fission yeast SMN1 gene
(B) Correct chromosomal integration of the tdSMN allele was checked by PCR amplification
using the indicated pairs of oligonuleotides and genomic DNA from tdSMN cells (td) or from
wild type cells (wt) as control.
Supplementary Figure 2
Splicing inhibition upon SMN depletion in S. pombe and separation of snRNPs on native gels
Splicing is inhibited in the tdSMN cells after shift to non-permissive temperature. After
growth of wild type cells and cells carrying the tdSMN allele at the indicated temperature
and time, total RNA was isolated and used for primer extension in the experiment shown in
panel A and for RT-PCR in the experiment shown in panel B. (A) Pre indicates the species
corresponding to the U6 precursor and mature indicates the spliced U6 RNA. (B) Splicing was
assayed for the portion of the cdc5 gene covering intron 4 and intron 5. The pre-mRNA and
mRNA species are schematized at the right. (C) Extracts prepared from tdSMN (td) and wild
type (wt) cells were fractionated on a 4% polyacrylamide gel together with 5 ug of wild-type
total RNA (RNA). The U1 RNA-containing complexes and free U1 snRNA were detected by
Northern hybridization using an oligonucleotide complementary to U1 RNA.
A
B
C
Supplementary Figure 3
Tiling array profiles of genes showing no major changes in introns and exons signals in the
tdSMN cells compared to wild type cells. The gene names and RT-PCR validation tests are
shown at right. See text for more details. Scale on the Y axis represents the log2 fold
change between the treatment (tdSMN) and control (wt) group signals.
Supplementary Figure 4
Primary sequences of the top 100 retained introns. The name of each gene is shown together
with the number of the retained intron. The branchpoint sequence is underlined. The first tract
of at least 5 contiguous pyrimidine located upstream or downstream from the branchpoint are
indicated in bold.
SPCC18B5.10c
I2
GTATGCAATGAAGTCTTTGACTGTTTGTTAACTATTTATTCAG
SPAC9.07c
I2
GTAAGTTTTCGAATTGCTTCTGCCGATCTTCGTGTTGCTAACTTCTTTAG
SPAC9G1.03c
I1
GTAAGTTTCATTCCAGTTTATGGCTTTATAAAATTGCTTTGTTCTAACTGAGTTATTAG
SPBC17D11.08
I3
GTATGCGATCCACAATCGGCAATTGGATTTTTCCTTTTGACAGTAGTTTGTTAACTTTCTTTAG
SPBC1711.04
I1
GTAAGTACCATCTTATTCAAAGATACACCATTACCGTTATAAAATAGAAATGAGGTTTGGCGAAGCCTTCTTCTT
TTTGTTTTTTTTCGAAATTTTTATTAATTGAATTTTTCAG
SPCC970.05
I1
GTATGTAAAATTTTGTTCTGTTACTGTACATGCTCGTAGAGAGCTTCAAAGCTTAGTATACTCAAATTCACTTGT
GGTATTTTGAGACTCGGAGCTATGAATACTGTAATGGGATATTATGTTTTGTTATTGAATTTATTCTTATTCTAA
CAACTTTTTAG
SPBC1685.09
I2
GTAAGAGAATTTTGCTGTAACTTTCTTTTAAGTTTGCAACATCTGTTTCCGACAGCATATTAACTAGTTTAG
SPAP27G11.03
I1
GTAAGTTAATATTTAAGTGCTTATGAACTAGTCCTTTGGAGTTATTTATTTTACTAACTTGAAAATAG
SPAPB18E9.01
I1
GTATGCTAAATTTTTCCATTAGGGTAATTTTGATATCATTTAACTAAATTTTTAG
SPBC13E7.09
I1
GTAAGTTTGCGATCATTAAAAGCATTATTTTTTTATTAACACAAAACTCTAG
SPBC106.18
I1
GTATGTTTTATGAATTTTCACGGAAAAAACGTCTCAGGTTCATTACAGTGTCCAAACATGGTGTCGTCAAAAGTT
CAGTGTATGGCATGATTTGCTAAGTAAATTACTTTTTACATTTGTAACAGAATATTACTTAAATTATTGCTGGAT
TTCCCGATACACTTTTTTATGGAGCTTAAGAACTTCGGTCTTTCCAAATATTTTTTAGATAAGAATATTTGCTAA
CTTATATCTTTATAG
SPAC17A5.07c
I3
GTAAGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGGAATCTTCGCTTCTTTTAACCTCAAAAATTTAG
SPBC11G11.02c
I1
GTACGGAAAACATGAGTTTGCGGTCTGAGTCGTTTAATCGAACGTGTGCTAATGTTACGAATATGGATAG
SPBC646.15c
I1
GTATGGATTTAGCTATTTGTTGTAATCTAACCTTTTGTTTAG
SPCC285.14
I4
GTATGTAAAAAAAAATATTTCACTATTGCTAACCTTATTTAG
SPAC4A8.07c
I2
GTTCGTTCTCTGTTTTCATTACATGTGTATATAATATGTATTAATTATTATTTATTACGATTTTTTTTAAAAAAC
TAACCACCTGAAAG
SPBC29A10.06c
I1
GTAAGTTGTCTTTAAAATGAAACTAAAAGAAAATATAATTTATTATGATTTTTATTTATTCATTAATTTATTATC
ATTCCATGAATCTTGATTGTTAACTCTGTTAG
SPBC336.08
I1
GTTAGTTTTAATCTAGAAATAAACGACCATGCATTAACTGTGAATAG
SPAC3A11.06
I7
GTAAGTATTCAAGTTGGTCACGTTTTGCTAATCCTTTTCTTAG
SPBC83.01
I2
GTATGAAAATGCGTTAATAGTTAGCTATATTCTAACATTAATCAG
SPBC660.16
I4
GTAATGATATTGATTGATATATTGAAGGGAAATATCGTATGCTATCCTGACAATTTGTATTGCCTTTTGCAGTAC
ATTCTTGTTGCGGAAAGTTCAATACTATTTTTTTCTTCATGGTTATCTTTCTTTCTTTTGAAATCTTTTTATTTG
GCTAACGGTTCTTTTTCTTCAG
SPBC215.02
I3
GTATGAGCATTTACAGTACAGATCAATTTTTATCTTATTATCAACAATTTGCTAATTCTCTGTAG
SPBC1539.06
I1
GTATTTTTTTCTTAACCCCCTTTTTTTGTAGCTTAGCTTACTAATTTGTTGTACTAG
SPBC2G2.10c
I1
GTAAGTTAATTAAATTGTTGAAATTTAAAGGTTAACATTTTAG
SPAC1705.02
I2
GTAAGTGGAATGGAATCAGGATTTTGTTTAAAAACCAATTAGCTAATATTTTAAG
SPAC23C11.10
I4
GTAAGTAAAGGTTCAAATAAGCGACAATTTTAACGCCTTCCAG
SPCC1393.06c
I2
GTATGAACAAAAGGAATTTTTATTCAAATAACTAACCTAAACATCTAG
SPBC106.15
I2
GTATGTGGTTGATCCGTGTTTCTCTTTATTGAACACTTGACTAACGGACATTCTAG
SPCC553.02
I1
GTATGCTATAATCATTTTTCGTCTCAATGATCTAACTTCTGTAG
SPAC26H5.13c
I1
GTATGTCTATTTTTTCCAGCTTTTTTCGATGTTTTAATCATGTATTTGTCATTTGCTCTTTTCTATTTTGATTTG
TACTAATTCATTAG
SPAP32A8.01c
I2
GTATGAAAGAATCATATCAAAAATTGTATTCTAACAACTTTGTTTTAG
SPAC24H6.03
I6
GTATGTCTTTTTCCTTTTTTTTTTTTTTCAAACATTTTTACTAACAGTAAATTAAG
SPAC22F3.14c
I1
GTAAGAAGTTCGAGGTAATACCGATTAATTTTTTATTAACAGAATATAATTTTAG
SPCC737.06c
I1
GTAAGCAAATGATGAATTGGACATTCAAACGAGTATCGTACTGCTTGGGTTTGGGCAGAAATATACTTGAATATT
TGTAGGCATAAATGTATTCTAACATATTTCTAG
SPCC24B10.17
I1
GTATGTAATTTCAGTGTATTTGTGTTGAGATTCATGGAGTATACAATGCTAATGAAGCAAACTTAG
SPAC1805.02c
I1
GTATGTTGAAAGGAGAGTAATCATTTCAGTACTGCTGTAATTCGCAATTTATATTTGACTAATCGTGAAAAG
SPCC4E9.01c
I3
GTGTGTGCATCATCTTTTTGTAATATTTACTTACCATACATATCGCTAG
SPBC13G1.11
I1
GTATGTGCAAGATTTAATTGAAGAGTAGTTTGTTAACAAATATTTCAG
SPCC1259.01c
I1
GTAAGTTTAAAGGCTAGCAACATGGTAAATTAATGTCACATTTACTAATTCTTAAATAG
SPAC15A10.13
I4
GTAAGCCATCTTATCTTACAAATTGCTAATTAGTATACAG
SPAC9E9.06c
I1
GTATGTCACAAATGCGTCTTACTTATAACTCGGTTTACTAATTGCCATTACTGTATAG
SPAC22F3.07c
I2
GTAAGCAACTATTTGCTACAAAGAACAGATCCACTAATTTCTCTTTTAG
SPCC24B10.17
I2
GTATGTGTTATCTTTTTGTTTTCTGGCATCAAAGCCTGGCATTATGGTCGTCATTTCCCATGTCGTCTAATTGTT
TGATGGTTGAGCCGGAAACAGCTAGATGTACAATCTTAACTAACAACATCAATAG
SPBC2A9.08c
I2
GTAAGTTTAATTGTGTATACGAATAGCAAACGGAATCTCAATTTTAGATCTAACCAAACGTCTAG
SPAC323.03c
I3
GTTAGTGTTTGACTTCACTGTTCTGGAAGACTAAATGATGCAG
SPAC3G9.09c
I2
GTAAGAATTTATTAGTAGTTTTTTTTTTTTTTTTTTTTAAAAAAAAGAAAAACAAAAGGGGAAAAAAGGTTTATA
AGTTGTTAGGAATGTACTAACCAGGATTAAG
SPBC1105.09
I1
GTAAGCGCACGAAATCTTCATATCCAAATATTCATGATTGTACATAGTATCTTAATTCTTGAACACATACTGATA
GATATAG
SPBC19C2.15c
I1
GTAAGACTCGATTTTTTATAGATCTCAGTTATTAACAAAGTTAAAAATAG
SPCC970.01
I7
GTAAGTCTGACTAAAACAATCTATGTCTGACAGCTTTTAG
SPBP35G2.05c
I2
GTATGTTTTTATTTTGCTACCCAAAACATATGGTCAAGACTTTGGTACTAACCGTATTTGCAAATAG
SPAP8A3.09c
I4
GTATGTCTGCTAAAAATAACTTTTCTTTTAAAAACCTCATTTTACTAATCGTTATATTTAG
SPAPB18E9.01
I6
GTATGAAATGCTTACTTTAAATGAAAATTTACTAACCAAGTTTTTTTAG
SPAC4D7.12c
I1
GTAAGTCACAGAATCATAATTCCTCGAGCTTCTGCTAATGACTAG
SPAC23A1.03
I1
GTAAGATTTTATAGAAGGAGGAAATGCGCGAGGTTGGATAGGAACGGTGATATTATTTTGCTCAATTAGCGTGAA
GTTTGAATCCAAAATACAATTCGAGAGGTTATATTATCTTTACGCCTTGCAATTGCTTAAATACTCTGTTAAATT
ATAAACAATAAGATGCTAATGTTATTAG
SPAC19B12.09
I2
GTATGTGCTTTAAAAGATTTTAAAGACGATTACTAATTTAAAAG
SPAC4G9.21c
I1
GTAAGTAATCGTTTTCTGTTTTATGGCATATACTAACTTTTCTTAG
SPBC839.09c
I1
GTACGAAGAAAATCCATTGAAGTTGCTATTGTCTTTCAAGAATATGATTTTCGATTGTTGGATTATTGTCTGCTA
ATACTTTATTGATATTTAG
SPAC29A4.14c
I2
GTATGTGTTGATAGTGCGTTGGCGGTAAATGTTTGAATCCGATTGGCGTTAATATATATGTGTTTTATCTTTCAT
CTTAATTTTTTAGTGCTAACACTTCGAACTAG
SPAC13A11.03
I1
GTAAGTTCGTGATATGATTGTTCAAGTGCCAAAACATGATTTTTGCCCATTCGTGTCTCACTTTCCACATTGCTA
ATCTATGTTTTTTTTATAG
SPAC17G6.06
I1
GTATGTATTTTTTTGCTTATAAGTTCTTTACGATCAGACGTGGCATATACTAACATGAGGGCGTTTTAG
SPBC211.05
I2
GTAAGTACTAATCGGTTTATTTGACAAATGAATTTTCGCTCACCATATAAACTAG
SPAC23C4.15
I2
GTATGTACATTTATTTAAATCCATCGTGATAATGGATTAAACAATCCGATTGATTATAGAAGCAAATTCTGTTGA
TATCTGCGTATATGTCCATAGACGCCGGTATTTATAAATCGGTTTACTCTTAATCTGCTTTCTAATTGATGAACA
TGCTTCTTCTAAACTTCCTTTATCAATTTTTTTTTAAAAACTGTTTTGCTAATGCGTTTAG
SPAC227.11c
I1
GTATGCTCTTGAACCTAATTTCTCTTCAATTCTAACGGACTTCCTTTGTAG
SPAC57A10.10c
I1
GTATGTTACACACTTTGTTTACGTTGGATTAAAACCTTTTTTGTGTTGAGTGGTCGTTACTAACAGTATGAATAG
SPAC31G5.17c
I1
GTATGTAATTTTTGGATGTGAAGTATTGTTATCGAGGGAGTCATAGAATGGATTAAAACGAAAAGGAGCTTTTCC
AAAGGTTTGATATCTTACAGAGCACATTTGAAATTAATAGCACAATATTTAAAAGTTGATTCAATTTCTGCTCAC
TCAACTATTGCATCTTTCGATAACTTGCTTGTCATACAGAATAGTTTCAAGGTGGCTAACCTTTAACAG
SPAC644.15
I1
GTACGTTATATTTTACTAGAAAAGTTGTGAATGTACTTCTACCGATACTGGTAAAAGATCTTTTCCATAGCGTGT
AGTCGTCTTATTCCTTCTTATTCGGTTTTTGTTAAATTAATTTATACTAATTGTTGCGTAG
SPAC1006.03c
I3
GTATTTGTGCTGTTGATTTTTGACTAACCTTTATACAG
SPBC947.14c
I1
GTATGCTTCTTATTACGTGCCTTGATAGGGTCGCCTTACCTTTGTTCGCCTTTTAACCAAATTGTAG
SPCC1259.05c
I1
GTAAGGAATGGTTTTGAATAACGCCGTATGTCTGAATCCGCTGAAACTGACGAATTTATTAG
SPAPB1A11.02
I1
GTGAGTTTTAAAAATGAGTACCAAATGTCTTAACATGACTTTGTTGTCACAGTTTGACTAAGGTCCGTTGTTTTC
GTTCATTGTTCTAACCTTGCAG
SPBC337.09
I2
GTATGTTGAATCAAATCAAACATTCAATTTCCAAAATCATCTGATATTGTCTAACTTCTATTAG
SPCC895.09c
I1
GTAAGTCAGCTCTTAAAAGATGCGAATTAACCAATCTATCGAACACATACTTACTAAAATCTTCTAAACAG
SPAC9.02c
I1
GTAAGTGGATTCTGAGACATTCTCATTGTTAGTTATCTTGTCAATGATTTGTCTACGCTTGTTTTTCGAATAGCC
AGATTAACTAAACGCTCAATAG
SPAC328.05
I6
GTATGTATATAAGGACGCTTTTGTGGGATTAGTTTTCCGCATCATTTTAAGTTTTTTTTGGAGATTACTGACTGA
ATATAG
SPBC776.05
I5
GTATGTCCATATTCGGTGTACTTAACTTTTCATGGCTAATACTAG
SPAC30D11.03
I2
GTATGTGACATTGAAATAAAAAAATATGTTTGCTAACATTATAG
SPCC1529.01
I3
GTAGGTAAAATAGTTATACTTTCTTCAATGAAACACCTCAACTTACCGTTATAG
SPBP16F5.02
I1
GTATGTTTAGACATGGTCGGATTTCCTTCAATTACTTGGAAACTGTACTCAATATAAACATTCCGACTTTACTCA
CAAAATTTTAG
SPBC530.13
I3
GTATGTTACAAGGTGCGCAATAATTGTTTTTTGTTTATAAGGAGACTTGCTTACAGGTGTATTAGCTAATCGAAG
AGAACAAG
SPAC17G8.04c
I2
GTATGTGCAAACTGTACAAACTGCTTAAAGACGGTTTATATCAATTACGTTTTTATTTATTGATTAAATTTACAA
TTCTTCTTGATTTCATTTTATTATGGTGTAAATGAATTTTAACTTTTTTAG
SPBC12D12.06
I1
GTAAGGTTATCAATTTTAATTTTAAAGGTTTTACGAGAAGGTAATTGGCCTTTAATTCATTCCTATTCTTCTTTA
AAATGTCTTAATTGAGTTTTGAATATCTGCTTTTCTATTTTATTACTGCTGAACATATTCTTGACAAATTGAGGC
TTGCGAGTGAGCTGATCTACGTCTAATTACTGATCAGAGGAATAGCTTTTCTGTCTACTTCTAGTCACGAGTATC
GACTAAACTGGATGTATCTTGTTTGAGTCATTTTGCGCAAATGTCTTGCAATCACAGTTTATTTCGGCTTCTCAT
TCGTTTCGTTGACTTTAATTTAAAGCGATTTTAGCCTCTTTGTTAGTTTTTGTTTAAAATATCTTCGAACCTTTA
TTATTCCTTAGTATACTTTTTCAAGCTGACCTTACAG
SPAC8F11.04
I1
GTAAGTATTTATTAATTAAATCTTTTTTTAAAAAATCTAACGATAAACATTTTTGTCGTTGCTCGGTTTAAAAAA
AGAATAGAAAAATTTTTTCGAAATATATGTGTATGATCTAACTGTTGTTCAG
SPCP31B10.08c
I1
GTAAGTGAAATTTATATTACAAGAAAAGCAAAGAGAAAAGGAGTTGATCATTGTCTGTACCGGTTGAAGTTTTTC
CACTTTCATTTGATTGATTTTCAAACAAAATATATAATTTATTTTGTTATTTCTATAATTTTTCTTGTTTTTATG
AGTTATTTCTTCGTTCTATTTTGCTAACTTATCGTTTTAG
SPBC1711.04
I2
GTAAGTGCCTCTAAAAATTTAACGTTCCTGCTTCTAATCATGAACAATTTAG
SPBP4H10.04
I2
GTATGTTGATTATCACCTTTCATATTTAGGCCCTAATTACTATAAGGTAAAATATTAGAACTTTGAAATGTAATT
TATGACTATGTTTTTCCTTCATTTTAATTTGGGTCGATATTTTGTTTGTATTGCAATATTCTTCGGTCTGACAGC
TTAG
SPBC3H7.01
I4
GTATGTGTTTACTTAAATATTTTTATTGCGATTTTGCCTTCTAATATGCATGCTAG
SPAC1834.12
I2
GTAAGTAATTGTGCGATTACTGCTCTTCATTATTAGACTTTTGCACAAAACACTTTTAACCATTAGTTATTATGA
TGAAAACAGTCTCTAAATCCTACGAAATGAAATTTATCTAACGTTAAATACTAG
SPBC21D10.05c
I2
GTAATTTTTTTTTAATTAATATTTTTCCTTTCAATTTTTAATAATTTTTTTTTTTACTAACGAGTATAG
SPAC16C9.02c
I1
GTATGATGAAGATTAACTGGAAGTTGGGATTTGCTAACGTTTTATTCAG
SPBC23E6.07c
I2
GTTCGTTTAATTCGGGTTATCAGTTTATATTTCTAACCCTCCAACTAG
SPBC1778.03c
I1
GTAAGTAGTTAATTGTTAGTTCTATTTAAAGCTACTTCAAACTTGATAATTCATATCATCTGTTTTGCATGCTCA
TGGAACGTACCGCTCAACCCTTGCTAATACATGTGCAATGTAACACATAAAATCAATTTTTGATATAATAATCTT
AAAAAAATGAGTTTTCTAACGTTTTTCAAG
SPAC2F3.12c
I1
GTAGGTTACAGTAGCGTACATATTATGTCTAATGTTGACATTAG
SPAC222.16c
I5
GTATCTTTTTTCCCTTGAAAAATCACAAGTACTAATTCTTTCGATTCAG
SPBC1539.05
I2
GTATTAGGAGCGTGCTGCATACCACTTATCATTACTAACACCTTGAACAG
SPCC18B5.10c
I5
GTATGTAGTTTCTTTTAAATTTTTTGGTCGCTAACTTCTAAAAAG
SPAC22F3.05c
I5
GTCAGTTTACAATTTATGAATCCAAGCTAACAACAAGTCAAG
SPBC1703.10
I4
GTATGTAATGTGTGAAGTATGGAAAACCAATTTGATTGTCTTCCGTTTTCTGTATATGCGCGTATATGCGTTATA
AGGCGTTTGGGGGATATTTTTATGGCGTTTTCTAGTCTTCACATTGTTTCCGACGCGGTTCCGAGGCTAATGCTT
GCTTCTTAG
SPCC1739.04c
I2
GTATGTGATTGTATCTTTATCTTCGTTTACGTACTAACCCTGTTGATACAG
SPBC16C6.10
I1
GTGCGCAACATTAATACTTGTCTTGCTAACCATGTAG
SPBC21C3.09c
I1
GTACGTTGTTACAAGTTCAAGATTTATTGATGATTCGAAGATATGGTTACTTGGAATCAAAAAAGCTGAAGGTCC
AAAACGTCACTGAATTGTCGAGGATCTTAAACTTTTATTTTACATTTGAAGTAGAATACTAATTTTATATTTAG
Supplementary Figure 5
Comparison of splicing consensus signals in defined groups of introns (A) Frequencies for 5'
splice sites, 3' acceptor sites and branch point sequences found in the defined groups of
retained introns compared to all fission yeast introns. (B) Histogram displaying the
percentage of pyrimidine tracts of 5 or more pyrimidines found in the 455 and 1827 groups
of retained introns in the tdSMN cells (tdSMN) compared to all S. pombe introns. Up: tracts
located at a maximum distance of 20 nucleotides from the branchpoint adenosine. Down:
pyrimidine tracts located immediately downstream of the branchpoint. Both: pyrimidine
tracts found upstream and downstream of the branch point.
Supplementary Figure 6
Primary sequences of the wild type reporter introns (WT) and the corresponding mutated
constructs (Mut). Mutations introduced to increase the length of PPT are shown in bold. The
name of each gene is shown together with the number of the retained intron.
SPBC106.18 Intron 1 WT
GTATGTTTTATGAATTTTCACGGAAAAAACGTCTCAGGTTCATTACAGTGTCCAAACATGGTGTCGTCAAAAGTTCAGTG
TATGGCATGATTTGCTAAGTAAATTACTTTTTACATTTGTAACAGAATATTACTTAAATTATTGCTGGATTTCCCGATAC
ACTTTTTTATGGAGCTTAAGAACTTCGGTCTTTCCAAATATTTTTTAGATAAGAATATTTGCTAACTTATATCTTTATAG
SPBC106.18 Intron 1 Mut
GTATGTTTTATGAATTTTCACGGAAAAAACGTCTCAGGTTCATTACAGTGTCCAAACATGGTGTCGTCAAAAGTTCAGTG
TATGGCATGATTTGCTAAGTAAATTACTTTTTACATTTGTAACAGAATATTACTTAAATTATTGCTGGATTTCCCGATAC
ACTTTTTTATGGAGCTTAAGAACTTCGGTCTTTCCAAATATTTTTTCTTTTTTCCTTTTTGCTAACTTATATCTTTATAG
SPBC947.14c Intron 1 WT
GTATGCTTCTTATTACGTGCCTTGATAGGGTCGCCTTACCTTTGTTCGCCTTTTAACCAAATTGTAG
SPBC947.14c Intron 1 Mut
GTATGCTTCTTATTACGTGCCTTGATAGGGTCGCCTTTCCTTTTTTCTCCTTTTAACCAAATTGTAG
SPAC630.03 Intron 3 WT
GTATGTATAAAAAATCTAATACGAGCTTAGATAAACAGTTTTGAACATTAAATGCATATATATATGCCCGAATTTTACTA
ACTGATAAACCTTAG
SPAC630.03 Intron 3 Mut
GTATGTATAAAAAATCTAATACGAGCTTAGATAAACAGTTTTGAACATTAAATGCATATATATATTCCCTTTTTTTACTA
ACTGATAAACCTTAG
SPAC4F10.14c Intron 1 WT
GTATGTTCGAATGCATCCCCGTGTTTTGAAGTTGACACCAGCTCTAAGAAATGCCTTGAACGGTTTCAAAGTGGTTCCAC
TGTAATTGATGCTGGCTATTGAAAACACAGTTGTACTTTTTTCATAGAGAACTGCTTAATTAACTTATCATTGTATAG
SPAC4F10.14c Intron 1 Mut
GTATGTTCGAATGCATCCCCGTGTTTTGAAGTTGACACCAGCTCTAAGAAATGCCTTGAACGGTTTCAAAGTGGTTCCAC
TGTAATTGATGCTGGCTATTGAAAACACAGTTGTACTTTTTTCTTTCTTTTCTTCTTAATTAACTTATCATTGTATAG
SPBC1703.10 Intron 4 WT
GTATGTAATGTGTGAAGTATGGAAAACCAATTTGATTGTCTTCCGTTTTCTGTATATGCGCGTATATGCGTTATAAGGCG
TTTGGGGGATATTTTTATGGCGTTTTCTAGTCTTCACATTGTTTCCGACGCGGTTCCGAGGCTAATGCTTGCTTCTTAG
SPBC1703.10 Intron 4 Mut
GTATGTAATGTGTGAAGTATGGAAAACCAATTTGATTGTCTTCCGTTTTCTGTATATGCGCGTATATGCGTTATAAGGCG
TTTGGGGGATATTTTTATGGCGTTTTCTAGTCTTCACATTGTTTCCGACTCTTTTCCTTTTCTAATGCTTGCTTCTTAG
SPCC24B10.17 Intron 2 WT
GTATGTGTTATCTTTTTGTTTTCTGGCATCAAAGCCTGGCATTATGGTCGTCATTTCCCATGTCGTCTAATTGTTTGATG
GTTGAGCCGGAAACAGCTAGATGTACAATCTTAACTAACAACATCAATAG
SPCC24B10.17 Intron 2 Mut
GTATGTGTTATCTTTTTGTTTTCTGGCATCAAAGCCTGGCATTATGGTCGTCATTTCCCATGTCGTCTAATTGTTTGATG
GTTGAGCCGGAAACAGCTTTTTTTCCTTTCTTAACTAACAACATCAATAG
SPAC9G1.03c Intron 1 WT
GTAAGTTTCATTCCAGTTTATGGCTTTATAAAATTGCTTTGTTCTAACTGAGTTATTAG
SPAC9G1.03c Intron 1 Mut
GTAAGTTTCATTCCAGTTTATGGCTTTCTTTCTTTTCTTTTTTCTAACTGAGTTATTAG
Supplementary Figure 7
Representative semi-quantitative RT-PCR experiment.
Following reverse-transcription of total RNA prepared from wild type (WT) and tdSMN cells
carrying the SPBC947.14c wild-type (I1) and mutated (I1 mut) reporters after growth at 25°C.
PCRs were carried out for the indicated number of cycles in the presence of 2 Ci
[32P]dCTP. The PCR products were separated on a non-denaturing 6%
polyacrylamide gel. The spliced and non-spliced species are schematized at the right.
Supplementary Table I
List of the 4614 introns from fission yeast sorted by their retention index (last column), which
correspond to the difference of the mean signals in the intron and the mean signal of the
adjacent exons. The rank of the intron is listed in the first column. The gene names and the
intron numbers are shown in columns n°2 and n°3, respectively. The start and end position of
the intron in the fission yeast Gene Database (www.genedb.org/genedb/pombe/index.jsp) are
also reported.
Supplementary Table II
List of the 622 introns from fission yeast sorted by their retention index. The rank of the
introns in the Supplementary Table I is shown in the second column. The gene names and the
intron numbers are indicated in columns n°3 and n°4, respectively. The other columns show
the sequence of the 5' donor, 3' acceptor and branchpoint sequences, respectively.
Supplementary Methods
In order to find common features of retained introns, we first used the non-parametric
Wilcoxon test (see Methods) to determine whether the ranked introns listed in supplementary
Table 1 could be divided into 2 groups significantly differing (p value < 0.05) by the
distribution of the sequences corresponding to the splicing signals used in S. pombe
(ftp://ftp.sanger.ac.uk/pub/yeast/pombe/Intron_Data/).
Since the majority of introns which are efficiently spliced in the tdSMN cells (Figure 5B) and
exhibit a low retention index contain long PPT (>=8 Pyr) located either immediately upstream
or downstream of the BP, we also analyzed the distribution of PPTs consisting of 5 or more
consecutive pyrimidines located immediately downstream of the BP or ending at most within
a 20 nucleotide distance upstream of the BP adenosine. Results are shown in Fig. 6A-C). For
the 1827 group, the frequency of PPTs at each position upstream of the branchpoint adenosine
was only slightly different from the remaining 2787 introns. Therefore, we also analyzed the
size distribution of the pyrimidine tracts (Fig. 6D).
Although these results indicate that the lack of a PPT located upstream of the branchpoint
might be a determinant accounting for introns retention in the tdSMN strain, it is not the only
one since introns containing fairly long PPTs located immediately upstream of the
branchpoint, as for exemple SPAC1952.08c I1 (rank 178 in Supplementary Table 1) and
SPAC3h8.03 I1 (rank 258), appear to be poorly spliced in the tdSMN cells. Since efficient
splicing obviously relies on the combinatorial effects of all splicing signals, it is possible that
suboptimal donor, acceptor and/or branchpoint sequences modulate the strength of the PPTs.
Therefore, analysis of PPT distribution was performed in the subset of S. pombe introns
containing the strongest consensus splice site. Results of this analysis are shown in Figure 7.