SMN deficiency in severe models of spinal muscular atrophy causes widespread intron retention and DNA damage

Edited by James L. Manley, Columbia University, New York, NY, and approved February 7, 2017 (received for review August 8, 2016)
March 7, 2017
114 (12) E2347-E2356

Significance

Spinal muscular atrophy is the leading monogenic cause of infant mortality and is caused by homozygous loss of the survival of motor neuron 1 (SMN1) gene. We investigated global transcriptome changes in the spinal cord of inducible SMA mice. SMN depletion caused widespread retention of introns with weak splice sites or belonging to the minor (U12) class. In addition, DNA double strand breaks accumulated in the spinal cord of SMA mice and in human SMA cell culture models. DNA damage was partially rescued by suppressing the formation of R-loops, which accumulated over retained introns. We propose that instead of single gene effects, pervasive splicing defects caused by severe SMN deficiency trigger a global DNA damage and stress response, thus compromising motor neuron survival.

Abstract

Spinal muscular atrophy (SMA), an autosomal recessive neuromuscular disease, is the leading monogenic cause of infant mortality. Homozygous loss of the gene survival of motor neuron 1 (SMN1) causes the selective degeneration of lower motor neurons and subsequent atrophy of proximal skeletal muscles. The SMN1 protein product, survival of motor neuron (SMN), is ubiquitously expressed and is a key factor in the assembly of the core splicing machinery. The molecular mechanisms by which disruption of the broad functions of SMN leads to neurodegeneration remain unclear. We used an antisense oligonucleotide (ASO)-based inducible mouse model of SMA to investigate the SMN-specific transcriptome changes associated with neurodegeneration. We found evidence of widespread intron retention, particularly of minor U12 introns, in the spinal cord of mice 30 d after SMA induction, which was then rescued by a therapeutic ASO. Intron retention was concomitant with a strong induction of the p53 pathway and DNA damage response, manifesting as γ-H2A.X positivity in neurons of the spinal cord and brain. Widespread intron retention and markers of the DNA damage response were also observed with SMN depletion in human SH-SY5Y neuroblastoma cells and human induced pluripotent stem cell-derived motor neurons. We also found that retained introns, high in GC content, served as substrates for the formation of transcriptional R-loops. We propose that defects in intron removal in SMA promote DNA damage in part through the formation of RNA:DNA hybrid structures, leading to motor neuron death.

Continue Reading

Data Availability

Data deposition: The sequence reported in this paper has been deposited in the Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE87281).

Acknowledgments

We thank Christopher Henderson for providing helpful guidance and discussion, and staff at Biogen Translational Pathology for conducting the immunohistochemistry studies. This work was supported in part by the St. Giles Foundation and the SMA Foundation (A.R.K.). Biogen and Ionis Pharmaceuticals provided funding for the acquisition, analysis, and interpretation of data. Biogen reviewed and provided feedback on the manuscript to the authors, and all authors provided their approval of the manuscript.

Supporting Information

Supporting Information (PDF)

References

1
K Talbot, KE Davies, Spinal muscular atrophy. Semin Neurol 21, 189–197 (2001).
2
DJ Birnkrant, JF Pope, JE Martin, AH Repucci, RM Eiben, Treatment of type I spinal muscular atrophy with noninvasive ventilation and gastrostomy feeding. Pediatr Neurol 18, 407–410 (1998).
3
L Campbell, A Potter, J Ignatius, V Dubowitz, K Davies, Genomic variation and gene conversion in spinal muscular atrophy: Implications for disease process and clinical phenotype. Am J Hum Genet 61, 40–50 (1997).
4
S Lefebvre, et al., Identification and characterization of a spinal muscular atrophy-determining gene. Cell 80, 155–165 (1995).
5
BG Burnett, et al., Regulation of SMN protein stability. Mol Cell Biol 29, 1107–1115 (2009).
6
JF Staropoli, et al., Rescue of gene-expression changes in an induced mouse model of spinal muscular atrophy by an antisense oligonucleotide that promotes inclusion of SMN2 exon 7. Genomics 105, 220–228 (2015).
7
K Sahashi, et al., Pathological impact of SMN2 mis-splicing in adult SMA mice. EMBO Mol Med 5, 1586–1601 (2013).
8
DD Coovert, et al., The survival motor neuron protein in spinal muscular atrophy. Hum Mol Genet 6, 1205–1214 (1997).
9
S Lefebvre, et al., Correlation between severity and SMN protein level in spinal muscular atrophy. Nat Genet 16, 265–269 (1997).
10
EF Tizzano, C Cabot, M Baiget, Cell-specific survival motor neuron gene expression during human development of the central nervous system: Implications for the pathogenesis of spinal muscular atrophy. Am J Pathol 153, 355–361 (1998).
11
MC Wahl, CL Will, R Lührmann, The spliceosome: Design principles of a dynamic RNP machine. Cell 136, 701–718 (2009).
12
AH Burghes, CE Beattie, Spinal muscular atrophy: Why do low levels of survival motor neuron protein make motor neurons sick? Nat Rev Neurosci 10, 597–609 (2009).
13
L Pellizzoni, Chaperoning ribonucleoprotein biogenesis in health and disease. EMBO Rep 8, 340–345 (2007).
14
DK Li, S Tisdale, F Lotti, L Pellizzoni, SMN control of RNP assembly: From post-transcriptional gene regulation to motor neuron disease. Semin Cell Dev Biol 32, 22–29 (2014).
15
L Pellizzoni, J Yong, G Dreyfuss, Essential role for the SMN complex in the specificity of snRNP assembly. Science 298, 1775–1779 (2002).
16
C Fallini, et al., The survival of motor neuron (SMN) protein interacts with the mRNA-binding protein HuD and regulates localization of poly(A) mRNA in primary motor neuron axons. J Neurosci 31, 3914–3925 (2011).
17
HL Zhang, et al., Active transport of the survival motor neuron protein and the role of exon-7 in cytoplasmic localization. J Neurosci 23, 6627–6637 (2003).
18
C Fallini, GJ Bassell, W Rossoll, Spinal muscular atrophy: The role of SMN in axonal mRNA regulation. Brain Res 1462, 81–92 (2012).
19
Z Zhang, et al., SMN deficiency causes tissue-specific perturbations in the repertoire of snRNAs and widespread defects in splicing. Cell 133, 585–600 (2008).
20
Z Zhang, et al., Dysregulation of synaptogenesis genes antecedes motor neuron pathology in spinal muscular atrophy. Proc Natl Acad Sci USA 110, 19348–19353 (2013).
21
EL Garcia, Z Lu, MP Meers, K Praveen, AG Matera, Developmental arrest of Drosophila survival motor neuron (Smn) mutants accounts for differences in expression of minor intron-containing genes. RNA 19, 1510–1516 (2013).
22
K Praveen, Y Wen, AG Matera, A Drosophila model of spinal muscular atrophy uncouples snRNP biogenesis functions of survival motor neuron from locomotion and viability defects. Cell Reports 1, 624–631 (2012).
23
D Bäumer, et al., Alternative splicing events are a late feature of pathology in a mouse model of spinal muscular atrophy. PLoS Genet 5, e1000773 (2009).
24
S Corti, et al., Neural stem cell transplantation can ameliorate the phenotype of a mouse model of spinal muscular atrophy. J Clin Invest 118, 3316–3330 (2008).
25
M Ruggiu, et al., A role for SMN exon 7 splicing in the selective vulnerability of motor neurons in spinal muscular atrophy. Mol Cell Biol 32, 126–138 (2012).
26
L Hubers, et al., HuD interacts with survival motor neuron protein and can rescue spinal muscular atrophy-like neuronal defects. Hum Mol Genet 20, 553–579 (2011).
27
H Tadesse, J Deschênes-Furry, S Boisvenue, J Côté, KH-type splicing regulatory protein interacts with survival motor neuron protein and is misregulated in spinal muscular atrophy. Hum Mol Genet 17, 506–524 (2008).
28
DY Zhao, et al., SMN and symmetric arginine dimethylation of RNA polymerase II C-terminal domain control termination. Nature 529, 48–53 (2016).
29
S Shen, et al., rMATS: robust and flexible detection of differential alternative splicing from replicate RNA-Seq data. Proc Natl Acad Sci USA 111, E5593–E5601 (2014).
30
TS Alioto, U12DB: A database of orthologous U12-type spliceosomal introns. Nucleic Acids Res 35, D110–D115 (2007).
31
Y Katz, ET Wang, EM Airoldi, CB Burge, Analysis and design of RNA sequencing experiments for identifying isoform regulation. Nat Methods 7, 1009–1015 (2010).
32
U Braunschweig, et al., Widespread intron retention in mammals functionally tunes transcriptomes. Genome Res 24, 1774–1786 (2014).
33
R Shalgi, JA Hurt, S Lindquist, CB Burge, Widespread inhibition of posttranscriptional splicing shapes the cellular transcriptome following heat shock. Cell Reports 7, 1362–1370 (2014).
34
JJ Wong, et al., Orchestrated intron retention regulates normal granulocyte differentiation. Cell 154, 583–595 (2013).
35
K Yap, ZQ Lim, P Khandelia, B Friedman, EV Makeyev, Coordinated regulation of neuronal mRNA steady-state levels through developmentally controlled intron retention. Genes Dev 26, 1209–1223 (2012).
36
G Yeo, CB Burge, Maximum entropy modeling of short sequence motifs with applications to RNA splicing signals. J Comput Biol 11, 377–394 (2004).
37
DS Park, et al., Cyclin-dependent kinases participate in death of neurons evoked by DNA-damaging agents. J Cell Biol 143, 457–467 (1998).
38
RD Paulsen, et al., A genome-wide siRNA screen reveals diverse cellular processes and pathways that mediate genome stability. Mol Cell 35, 228–239 (2009).
39
A Aguilera, T García-Muse, R loops: From transcription byproducts to threats to genome stability. Mol Cell 46, 115–124 (2012).
40
X Li, JL Manley, Cotranscriptional processes and their influence on genome stability. Genes Dev 20, 1838–1847 (2006).
41
ML Duquette, P Handa, JA Vincent, AF Taylor, N Maizels, Intracellular transcription of G-rich DNAs induces formation of G-loops, novel structures containing G4 DNA. Genes Dev 18, 1618–1629 (2004).
42
MM Denis, et al., Escaping the nuclear confines: Signal-dependent pre-mRNA splicing in anucleate platelets. Cell 122, 379–391 (2005).
43
MS Domínguez-Sánchez, S Barroso, B Gómez-González, R Luna, A Aguilera, Genome instability and transcription elongation impairment in human cells depleted of THO/TREX. PLoS Genet 7, e1002386 (2011).
44
PA Ginno, PL Lott, HC Christensen, I Korf, F Chédin, R-loop formation is a distinctive characteristic of unmethylated human CpG island promoters. Mol Cell 45, 814–825 (2012).
45
K Skourti-Stathaki, NJ Proudfoot, N Gromak, Human senataxin resolves RNA/DNA hybrids formed at transcriptional pause sites to promote Xrn2-dependent termination. Mol Cell 42, 794–805 (2011).
46
ZF Chou, F Chen, J Wilusz, Sequence and position requirements for uridylate-rich downstream elements of polyadenylation signals. Nucleic Acids Res 22, 2525–2531 (1994).
47
KA Montzka, JA Steitz, Additional low-abundance human small nuclear ribonucleoproteins: U11, U12, etc. Proc Natl Acad Sci USA 85, 8885–8889 (1988).
48
F Lotti, et al., An SMN-dependent U12 splicing event essential for motor circuit function. Cell 151, 440–454 (2012).
49
Y Jia, JC Mu, SL Ackerman, Mutation of a U2 snRNA gene causes global disruption of alternative splicing and neurodegeneration. Cell 148, 296–308 (2012).
50
R Madabhushi, L Pan, LH Tsai, DNA damage and its links to neurodegeneration. Neuron 83, 266–282 (2014).
51
Y Shiloh, Y Ziv, The ATM protein kinase: Regulating the cellular response to genotoxic stress, and more. Nat Rev Mol Cell Biol 14, 197–210 (2013).
52
H Date, et al., Early-onset ataxia with ocular motor apraxia and hypoalbuminemia is caused by mutations in a new HIT superfamily gene. Nat Genet 29, 184–188 (2001).
53
MC Moreira, et al., The gene mutated in ataxia-ocular apraxia 1 encodes the new HIT/Zn-finger protein aprataxin. Nat Genet 29, 189–193 (2001).
54
LH Sanders, et al., Mitochondrial DNA damage: Molecular marker of vulnerable nigral neurons in Parkinson’s disease. Neurobiol Dis 70, 214–223 (2014).
55
MA Lovell, WR Markesbery, Oxidative DNA damage in mild cognitive impairment and late-stage Alzheimer’s disease. Nucleic Acids Res 35, 7497–7504 (2007).
56
L Wahba, JD Amon, D Koshland, M Vuica-Ross, RNase H and multiple RNA biogenesis factors cooperate to prevent RNA:DNA hybrids from generating genome instability. Mol Cell 44, 978–988 (2011).
57
AR Haeusler, et al., C9orf72 nucleotide repeat structures initiate molecular cascades of disease. Nature 507, 195–200 (2014).
58
E Grabczyk, M Mancuso, MC Sammarco, A persistent RNA.DNA hybrid formed by transcription of the Friedreich ataxia triplet repeat in live bacteria, and by T7 RNAP in vitro. Nucleic Acids Res 35, 5351–5359 (2007).
59
YZ Chen, et al., DNA/RNA helicase gene mutations in a form of juvenile amyotrophic lateral sclerosis (ALS4). Am J Hum Genet 74, 1128–1135 (2004).
60
K Grohmann, et al., Mutations in the gene encoding immunoglobulin mu-binding protein 2 cause spinal muscular atrophy with respiratory distress type 1. Nat Genet 29, 75–77 (2001).
61
Y Fukita, et al., The human S mu bp-2, a DNA-binding protein specific to the single-stranded guanine-rich sequence related to the immunoglobulin mu chain switch region. J Biol Chem 268, 17463–17470 (1993).
62
SC Ling, M Polymenidou, DW Cleveland, Converging mechanisms in ALS and FTD: Disrupted RNA and protein homeostasis. Neuron 79, 416–438 (2013).
63
C Lagier-Tourenne, DW Cleveland, Rethinking ALS: The FUS about TDP-43. Cell 136, 1001–1004 (2009).
64
H Han, et al., MBNL proteins repress ES-cell-specific alternative splicing and reprogramming. Nature 498, 241–245 (2013).
65
S Sun, et al., ALS-causative mutations in FUS/TLS confer gain and loss of function by altered association with SMN and U1-snRNP. Nat Commun 6, 6171 (2015).
66
Y Hua, J Zhou, Survival motor neuron protein facilitates assembly of stress granules. FEBS Lett 572, 69–74 (2004).
67
JS Salvi, K Mekhail, R-loops highlight the nucleus in ALS. Nucleus 6, 23–29 (2015).
68
SJ Hill, et al., Two familial ALS proteins function in prevention/repair of transcription-associated DNA damage. Proc Natl Acad Sci USA 113, E7701–E7709 (2016).
69
EL Garcia, Y Wen, K Praveen, AG Matera, Transcriptomic comparison of Drosophila snRNP biogenesis mutants reveals mutant-specific changes in pre-mRNA processing: Implications for spinal muscular atrophy. RNA 22, 1215–1227 (2016).
70
S Fayzullina, LJ Martin, Skeletal muscle DNA damage precedes spinal motor neuron DNA damage in a mouse model of spinal muscular atrophy (SMA). PLoS One 9, e93329 (2014).
71
EW Loomis, LA Sanz, F Chedin, PJ Hagerman, Transcription-associated R-loop formation across the human FMR1 CGG-repeat region. PLoS Genet 10, e1004294 (2014).
72
Y Maury, et al., Combinatorial analysis of developmental cues efficiently converts human pluripotent stem cells into multiple neuronal subtypes. Nat Biotechnol 33, 89–96 (2014).
73
AR Quinlan, IM Hall, BEDTools: A flexible suite of utilities for comparing genomic features. Bioinformatics 26, 841–842 (2010).

Information & Authors

Information

Published in

Go to Proceedings of the National Academy of Sciences
Go to Proceedings of the National Academy of Sciences
Proceedings of the National Academy of Sciences
Vol. 114 | No. 12
March 21, 2017
PubMed: 28270613

Classifications

Data Availability

Data deposition: The sequence reported in this paper has been deposited in the Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE87281).

Submission history

Published online: March 7, 2017
Published in issue: March 21, 2017

Keywords

  1. SMA
  2. SMN
  3. DNA damage
  4. neurodegeneration
  5. splicing

Acknowledgments

We thank Christopher Henderson for providing helpful guidance and discussion, and staff at Biogen Translational Pathology for conducting the immunohistochemistry studies. This work was supported in part by the St. Giles Foundation and the SMA Foundation (A.R.K.). Biogen and Ionis Pharmaceuticals provided funding for the acquisition, analysis, and interpretation of data. Biogen reviewed and provided feedback on the manuscript to the authors, and all authors provided their approval of the manuscript.

Notes

This article is a PNAS Direct Submission.

Authors

Affiliations

Mohini Jangi
Computational Biology & Genomics, Biogen, Cambridge, MA 02142;
Present address: GRAIL, Inc., Menlo Park, CA 94025.
Christina Fleet
Computational Biology & Genomics, Biogen, Cambridge, MA 02142;
Patrick Cullen
Computational Biology & Genomics, Biogen, Cambridge, MA 02142;
Shipra V. Gupta
Computational Biology & Genomics, Biogen, Cambridge, MA 02142;
Shila Mekhoubad
Stem Cell Research, Biogen, Cambridge, MA 02142;
Eric Chiao
Stem Cell Research, Biogen, Cambridge, MA 02142;
Present address: Cell Technologies, Regeneron Pharmaceuticals, Tarrytown, NY 10591
Norm Allaire
Computational Biology & Genomics, Biogen, Cambridge, MA 02142;
C. Frank Bennett
Neuroscience Drug Discovery, Ionis Pharmaceuticals, Carlsbad, CA 92008;
Frank Rigo
Neuroscience Drug Discovery, Ionis Pharmaceuticals, Carlsbad, CA 92008;
Adrian R. Krainer
Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724;
Jessica A. Hurt
Computational Biology & Genomics, Biogen, Cambridge, MA 02142;
John P. Carulli3 [email protected]
Computational Biology & Genomics, Biogen, Cambridge, MA 02142;
John F. Staropoli3 [email protected]
Rare Disease, Biogen, Cambridge, MA 02142

Notes

3
To whom correspondence may be addressed. Email: [email protected] or [email protected].
Author contributions: M.J., J.P.C., and J.F.S. designed research; M.J. and C.F. performed research; P.C., S.V.G., S.M., E.C., N.A., C.F.B., F.R., A.R.K., and J.A.H. contributed new reagents/analytic tools; M.J., J.A.H., J.P.C., and J.F.S. analyzed data; and M.J. wrote the paper.

Competing Interests

Conflict of interest statement: C.F., P.C., S.V.G., S.M., N.A., J.A.H., J.P.C., and J.F.S. are employees of Biogen. M.J. and E.C. were employed at Biogen at the time the work was performed, but are now employed at Grail and Regeneron, respectively. C.F.B. and F.R. are employees of Ionis Pharmaceuticals. A.R.K. serves as a consultant to Ionis Pharmaceuticals.

Metrics & Citations

Metrics

Note: The article usage is presented with a three- to four-day delay and will update daily once available. Due to ths delay, usage data will not appear immediately following publication. Citation information is sourced from Crossref Cited-by service.


Citation statements

Altmetrics

Citations

If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download.

Cited by

    Loading...

    View Options

    View options

    PDF format

    Download this article as a PDF file

    DOWNLOAD PDF

    Get Access

    Login options

    Check if you have access through your login credentials or your institution to get full access on this article.

    Personal login Institutional Login

    Recommend to a librarian

    Recommend PNAS to a Librarian

    Purchase options

    Purchase this article to get full access to it.

    Single Article Purchase

    SMN deficiency in severe models of spinal muscular atrophy causes widespread intron retention and DNA damage
    Proceedings of the National Academy of Sciences
    • Vol. 114
    • No. 12
    • pp. 2995-E2544

    Media

    Figures

    Tables

    Other

    Share

    Share

    Share article link

    Share on social media