Skip to main content

Transcriptional Elongation Regulator 1 Affects Transcription and Splicing of Genes Associated with Cellular Morphology and Cytoskeleton Dynamics and Is Required for Neurite Outgrowth in Neuroblastoma Cells and Primary Neuronal Cultures

Abstract

TCERG1 is a highly conserved human protein implicated in interactions with the transcriptional and splicing machinery that is associated with neurodegenerative disorders. Biochemical, neuropathological, and genetic evidence suggests an important role for TCERG1 in Huntington’s disease (HD) pathogenesis. At present, the molecular mechanism underlying TCERG1-mediated neuronal effects is unknown. Here, we show that TCERG1 depletion led to widespread alterations in mRNA processing that affected different types of alternative transcriptional or splicing events, indicating that TCERG1 plays a broad role in the regulation of alternative splicing. We observed considerable changes in the transcription and alternative splicing patterns of genes involved in cytoskeleton dynamics and neurite outgrowth. Accordingly, TCERG1 depletion in the neuroblastoma SH-SY5Y cell line and primary mouse neurons affected morphogenesis and resulted in reduced dendritic outgrowth, with a major effect on dendrite ramification and branching complexity. These defects could be rescued by ectopic expression of TCERG1. Our results indicate that TCERG1 affects expression of multiple mRNAs involved in neuron projection development, whose misregulation may be involved in TCERG1-linked neurological disorders.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Abbreviations

TCERG1:

Transcription elongation regulator 1

HD:

Huntington’s disease

RNAPII:

RNA polymerase II;

qPCR:

Quantitative PCR

References

  1. 1.

    Pan Q, Shai O, Lee LJ, Frey BJ, Blencowe BJ (2008) Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing. Nat Genet 40:1413–1415

    CAS  Article  PubMed  Google Scholar 

  2. 2.

    Wang ET, Sandberg R, Luo S, Khrebtukova I, Zhang L, Mayr C et al (2008) Alternative isoform regulation in human tissue transcriptomes. Nature 456:470–476

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  3. 3.

    Calarco JA, Zhen M, Blencowe BJ (2011) Networking in a global world: establishing functional connections between neural splicing regulators and their target transcripts. RNA 17:775–791

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  4. 4.

    Kelemen O, Convertini P, Zhang Z, Wen Y, Shen M, Falaleeva M et al (2013) Function of alternative splicing. Gene 514:1–30

    CAS  Article  PubMed  Google Scholar 

  5. 5.

    Licatalosi DD, Darnell RBRNA Processing and its regulation: global insights into biological networks. Nat Rev Genet 11:75–87

  6. 6.

    Singh RK, Cooper TA (2012) Pre-mRNA splicing in disease and therapeutics. Trends Mol Med 18:472–482

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  7. 7.

    Cooper TA, Wan L, Dreyfuss G (2009) RNA and disease. Cell 136:777–793

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  8. 8.

    Kulkarni VA, Firestein BL (2012) The dendritic tree and brain disorders. Mol Cell Neurosci 50:10–20

    CAS  Article  PubMed  Google Scholar 

  9. 9.

    Lenzken SC, Achsel T, Carri MT, Barabino SM (2014) Neuronal RNA-binding proteins in health and disease. Wiley Interdiscip Rev RNA 5:565–576

    CAS  Article  PubMed  Google Scholar 

  10. 10.

    Ward AJ, Cooper TA (2010) The pathobiology of splicing. J Pathol 220:152–163

    CAS  PubMed  PubMed Central  Google Scholar 

  11. 11.

    Suñé C, Hayashi T, Liu Y, Lane WS, Young RA, Garcia-Blanco MA (1997) CA150, a nuclear protein associated with the RNA polymerase II holoenzyme, is involved in tat-activated human immunodeficiency virus type 1 transcription. Mol Cell Biol 17:6029–6039

    Article  PubMed  PubMed Central  Google Scholar 

  12. 12.

    Goldstrohm AC, Albrecht TR, Suñé C, Bedford MT, Garcia-Blanco MA (2001) The transcription elongation factor CA150 interacts with RNA polymerase II and the pre-mRNA splicing factor SF1. Mol Cell Biol 21:7617–7628

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  13. 13.

    Lin KT, Lu RM, Tarn WY (2004) The WW domain-containing proteins interact with the early spliceosome and participate in pre-mRNA splicing in vivo. Mol Cell Biol 24:9176–9185

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  14. 14.

    Sánchez-Álvarez M, Goldstrohm AC, Garcia-Blanco MA, Suñé C (2006) Human transcription elongation factor CA150 localizes to splicing factor-rich nuclear speckles and assembles transcription and splicing components into complexes through its amino and carboxyl regions. Mol Cell Biol 26:4998–5014

    Article  PubMed  PubMed Central  Google Scholar 

  15. 15.

    Carty SM, Goldstrohm AC, Sune C, Garcia-Blanco MA, Greenleaf AL (2000) Protein-interaction modules that organize nuclear function: FF domains of CA150 bind the phosphoCTD of RNA polymerase II. Proc Natl Acad Sci U S A 97:9015–9020

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  16. 16.

    Smith MJ, Kulkarni S, Pawson T (2004) FF domains of CA150 bind transcription and splicing factors through multiple weak interactions. Mol Cell Biol 24:9274–9285

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  17. 17.

    Sánchez-Hernández N, Ruiz L, Sánchez-Álvarez M, Montes M, Macias MJ, Hernández-Munain C et al (2012) The FF4 and FF5 domains of transcription elongation regulator 1 (TCERG1) target proteins to the periphery of speckles. J Biol Chem 287:17789–17800

    Article  PubMed  PubMed Central  Google Scholar 

  18. 18.

    McFie PJ, Wang GL, Timchenko NA, Wilson HL, Hu X, Roesler WJ (2006) Identification of a co-repressor that inhibits the transcriptional and growth-arrest activities of CCAAT/enhancer-binding protein alpha. J Biol Chem 281:18069–18080

    CAS  Article  PubMed  Google Scholar 

  19. 19.

    Cheng D, Cote J, Shaaban S, Bedford MT (2007) The arginine methyltransferase CARM1 regulates the coupling of transcription and mRNA processing. Mol Cell 25:71–83

    Article  PubMed  Google Scholar 

  20. 20.

    Pearson JL, Robinson TJ, Munoz MJ, Kornblihtt AR, Garcia-Blanco MA (2008) Identification of the cellular targets of the transcription factor TCERG1 reveals a prevalent role in mRNA processing. J Biol Chem 283:7949–7961

    CAS  Article  PubMed  Google Scholar 

  21. 21.

    Sánchez-Álvarez M, Montes M, Sánchez-Hernández N, Hernández-Munain C, Suñé C (2010) Differential effects of sumoylation on transcription and alternative splicing by transcription elongation regulator 1 (TCERG1). J Biol Chem 285:15220–15233

    Article  PubMed  PubMed Central  Google Scholar 

  22. 22.

    Montes M, Cloutier A, Sánchez-Hernández N, Michelle L, Lemieux B, Blanchette M et al (2012) TCERG1 regulates alternative splicing of Bcl-x gene by modulating the rate of RNAPII transcription. Mol Cell Biol 32:751–762

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  23. 23.

    Goldstrohm AC, Greenleaf AL, Garcia-Blanco MA (2001) Co-transcriptional splicing of pre-messenger RNAs: considerations for the mechanism of alternative splicing. Gene 277:31–47

    CAS  Article  PubMed  Google Scholar 

  24. 24.

    Liu J, Fan S, Lee CJ, Greenleaf AL, Zhou P (2013) Specific interaction of the transcription elongation regulator TCERG1 with RNA polymerase II requires simultaneous phosphorylation at Ser2, Ser5, and Ser7 within the carboxyl-terminal domain repeat. J Biol Chem 288:10890–10901

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  25. 25.

    Holbert S, Denghien I, Kiechle T, Rosenblatt A, Wellington C, Hayden MR et al (2001) The Gln-Ala repeat transcriptional activator CA150 interacts with huntingtin: neuropathologic and genetic evidence for a role in Huntington’s disease pathogenesis. Proc Natl Acad Sci U S A 98:1811–1816

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  26. 26.

    Arango M, Holbert S, Zala D, Brouillet E, Pearson J, Regulier E et al (2006) CA150 expression delays striatal cell death in overexpression and knock-in conditions for mutant huntingtin neurotoxicity. J Neurosci 26:4649–4659

    CAS  Article  PubMed  Google Scholar 

  27. 27.

    de la Grange P, Gratadou L, Delord M, Dutertre M, Auboeuf D (2010) Splicing factor and exon profiling across human tissues. Nucleic Acids Res 38:2825–2838

    Article  PubMed  PubMed Central  Google Scholar 

  28. 28.

    Coiras M, Montes M, Montanuy I, Lopez-Huertas MR, Mateos E, Le Sommer C et al (2013) Transcription elongation regulator 1 (TCERG1) regulates competent RNA polymerase II-mediated elongation of HIV-1 transcription and facilitates efficient viral replication. Retrovirology 10:124

    Article  PubMed  PubMed Central  Google Scholar 

  29. 29.

    Pedraza N, Ortiz R, Cornado A, Llobet A, Aldea M, Gallego C (2014) KIS, a kinase associated with microtubule regulators, enhances translation of AMPA receptors and stimulates dendritic spine remodeling. J Neurosci 34:13988–13997

    Article  PubMed  Google Scholar 

  30. 30.

    Suñé C, Garcia-Blanco MA (1999) Transcriptional cofactor CA150 regulates RNA polymerase II elongation in a TATA-box-dependent manner. Mol Cell Biol 19:4719–4728

    Article  PubMed  PubMed Central  Google Scholar 

  31. 31.

    de la Grange P, Dutertre M, Correa M, Auboeuf D (2007) A new advance in alternative splicing databases: from catalogue to detailed analysis of regulation of expression and function of human alternative splicing variants. BMC Bioinformatics 8:180

    Article  PubMed  PubMed Central  Google Scholar 

  32. 32.

    de la Grange P, Dutertre M, Martin N, Auboeuf D (2005) FAST DB: a website resource for the study of the expression regulation of human gene products. Nucleic Acids Res 33:4276–4284

    Article  PubMed  PubMed Central  Google Scholar 

  33. 33.

    Huang da W, Sherman BT, Lempicki RA (2009) Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc 4:44–57

    Article  PubMed  Google Scholar 

  34. 34.

    Huang da W, Sherman BT, Lempicki RA (2009) Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists. Nucleic Acids Res 37:1–13

    Article  PubMed  Google Scholar 

  35. 35.

    Untergasser A, Nijveen H, Rao X, Bisseling T, Geurts R, Leunissen JA (2007) Primer3Plus, an enhanced web interface to Primer3. Nucleic Acids Res 35:W71–W74

    Article  PubMed  PubMed Central  Google Scholar 

  36. 36.

    Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc 3:1101–1108

    CAS  Article  PubMed  Google Scholar 

  37. 37.

    Wearne SL, Rodriguez A, Ehlenberger DB, Rocher AB, Henderson SC, Hof PR (2005) New techniques for imaging, digitization and analysis of three-dimensional neural morphology on multiple scales. Neuroscience 136:661–680

    CAS  Article  PubMed  Google Scholar 

  38. 38.

    Gardina PJ, Clark TA, Shimada B, Staples MK, Yang Q, Veitch J et al (2006) Alternative splicing and differential gene expression in colon cancer detected by a whole genome exon array. BMC Genomics 7:325

    Article  PubMed  PubMed Central  Google Scholar 

  39. 39.

    Amit M, Donyo M, Hollander D, Goren A, Kim E, Gelfman S et al (2012) Differential GC content between exons and introns establishes distinct strategies of splice-site recognition. Cell Rep 1:543–556

    CAS  Article  PubMed  Google Scholar 

  40. 40.

    Jonkers I, Kwak H, Lis JT (2014) Genome-wide dynamics of Pol II elongation and its interplay with promoter proximal pausing, chromatin, and exons. Elife 3:e02407

    Article  PubMed  PubMed Central  Google Scholar 

  41. 41.

    Veloso A, Kirkconnell KS, Magnuson B, Biewen B, Paulsen MT, Wilson TE et al (2014) Rate of elongation by RNA polymerase II is associated with specific gene features and epigenetic modifications. Genome Res 24:896–905

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  42. 42.

    Yeo GW, Coufal NG, Liang TY, Peng GE, Fu XD, Gage FH (2009) An RNA code for the FOX2 splicing regulator revealed by mapping RNA-protein interactions in stem cells. Nat Struct Mol Biol 16:130–137

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  43. 43.

    Benita Y, Cao Z, Giallourakis C, Li C, Gardet A, Xavier RJ (2010) Gene enrichment profiles reveal T-cell development, differentiation, and lineage-specific transcription factors including ZBTB25 as a novel NF-AT repressor. Blood 115:5376–5384

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  44. 44.

    Graham FL, Smiley J, Russell WC, Nairn R (1977) Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J Gen Virol 36:59–74

    CAS  Article  PubMed  Google Scholar 

  45. 45.

    Shaw G, Morse S, Ararat M, Graham FL (2002) Preferential transformation of human neuronal cells by human adenoviruses and the origin of HEK 293 cells. FASEB J 16:869–871

    CAS  PubMed  Google Scholar 

  46. 46.

    He B, Soderlund DM (2010) Human embryonic kidney (HEK293) cells express endogenous voltage-gated sodium currents and Na v 1.7 sodium channels. Neurosci Lett 469:268–272

    CAS  Article  PubMed  Google Scholar 

  47. 47.

    Vetter I, Lewis RJ (2010) Characterization of endogenous calcium responses in neuronal cell lines. Biochem Pharmacol 79:908–920

    CAS  Article  PubMed  Google Scholar 

  48. 48.

    Fu Z, Washbourne P, Ortinski P, Vicini S (2003) Functional excitatory synapses in HEK293 cells expressing neuroligin and glutamate receptors. J Neurophysiol 90:3950–3957

    CAS  Article  PubMed  Google Scholar 

  49. 49.

    Kim S, Burette A, Chung HS, Kwon SK, Woo J, Lee HW et al (2006) NGL family PSD-95-interacting adhesion molecules regulate excitatory synapse formation. Nat Neurosci 9:1294–1301

    CAS  Article  PubMed  Google Scholar 

  50. 50.

    Mah W, Ko J, Nam J, Han K, Chung WS, Kim E (2010) Selected SALM (synaptic adhesion-like molecule) family proteins regulate synapse formation. J Neurosci 30:5559–5568

    CAS  Article  PubMed  Google Scholar 

  51. 51.

    Woo J, Kwon SK, Choi S, Kim S, Lee JR, Dunah AW et al (2009) Trans-synaptic adhesion between NGL-3 and LAR regulates the formation of excitatory synapses. Nat Neurosci 12:428–437

    CAS  Article  PubMed  Google Scholar 

  52. 52.

    Campbell SA, Lin J, Dobrikova EY, Gromeier M (2005) Genetic determinants of cell type-specific poliovirus propagation in HEK 293 cells. J Virol 79:6281–6290

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  53. 53.

    Jahan N, Wimmer E, Mueller S (2011) A host-specific, temperature-sensitive translation defect determines the attenuation phenotype of a human rhinovirus/poliovirus chimera, PV1(RIPO). J Virol 85:7225–7235

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  54. 54.

    Madhusudana SN, Sundaramoorthy S, Ullas PT (2010) Utility of human embryonic kidney cell line HEK-293 for rapid isolation of fixed and street rabies viruses: comparison with Neuro-2a and BHK-21 cell lines. Int J Infect Dis 14:e1067–e1071

    CAS  Article  PubMed  Google Scholar 

  55. 55.

    Pahlman S, Hoehner JC, Nanberg E, Hedborg F, Fagerstrom S, Gestblom C et al (1995) Differentiation and survival influences of growth factors in human neuroblastoma. Eur J Cancer 31A:453–458

    CAS  Article  PubMed  Google Scholar 

  56. 56.

    Hotulainen P, Hoogenraad CC (2010) Actin in dendritic spines: connecting dynamics to function. J Cell Biol 189:619–629

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  57. 57.

    McGlincy NJ, Smith CW (2008) Alternative splicing resulting in nonsense-mediated mRNA decay: what is the meaning of nonsense? Trends Biochem Sci 33:385–393

    CAS  Article  PubMed  Google Scholar 

  58. 58.

    Jia J, Yao P, Arif A, Fox PL (2013) Regulation and dysregulation of 3’UTR-mediated translational control. Curr Opin Genet Dev 23:29–34

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  59. 59.

    Ha K, Coulombe-Huntington J, Majewski J (2009) Comparison of affymetrix gene array with the exon array shows potential application for detection of transcript isoform variation. BMC Genomics 10:519

    Article  PubMed  PubMed Central  Google Scholar 

  60. 60.

    Parrish JZ, Emoto K, Kim MD, Jan YN (2007) Mechanisms that regulate establishment, maintenance, and remodeling of dendritic fields. Annu Rev Neurosci 30:399–423

    CAS  Article  PubMed  Google Scholar 

  61. 61.

    Kasai H, Fukuda M, Watanabe S, Hayashi-Takagi A, Noguchi J (2010) Structural dynamics of dendritic spines in memory and cognition. Trends Neurosci 33:121–129

    CAS  Article  PubMed  Google Scholar 

  62. 62.

    Shirao T, Gonzalez-Billault C (2013) Actin filaments and microtubules in dendritic spines. J Neurochem 126:155–164

    CAS  Article  PubMed  Google Scholar 

  63. 63.

    Luo L (2002) Actin cytoskeleton regulation in neuronal morphogenesis and structural plasticity. Annu Rev Cell Dev Biol 18:601–635

    CAS  Article  PubMed  Google Scholar 

  64. 64.

    Cambray S, Pedraza N, Rafel M, Gari E, Aldea M, Gallego C (2009) Protein kinase KIS localizes to RNA granules and enhances local translation. Mol Cell Biol 29:726–735

    CAS  Article  PubMed  Google Scholar 

  65. 65.

    Jaworski J, Kapitein LC, Gouveia SM, Dortland BR, Wulf PS, Grigoriev I et al (2009) Dynamic microtubules regulate dendritic spine morphology and synaptic plasticity. Neuron 61:85–100

    CAS  Article  PubMed  Google Scholar 

  66. 66.

    Andresen JM, Gayan J, Cherny SS, Brocklebank D, Alkorta-Aranburu G, Addis EA et al (2007) Replication of twelve association studies for Huntington’s disease residual age of onset in large Venezuelan kindreds. J Med Genet 44:44–50

    CAS  Article  PubMed  Google Scholar 

  67. 67.

    Chattopadhyay B, Ghosh S, Gangopadhyay PK, Das SK, Roy T, Sinha KK et al (2003) Modulation of age at onset in Huntington’s disease and spinocerebellar ataxia type 2 patients originated from eastern India. Neurosci Lett 345:93–96

    CAS  Article  PubMed  Google Scholar 

  68. 68.

    Dunah AW, Jeong H, Griffin A, Kim YM, Standaert DG, Hersch SM et al (2002) Sp1 and TAFII130 transcriptional activity disrupted in early Huntington’s disease. Science 296:2238–2243

    CAS  Article  PubMed  Google Scholar 

  69. 69.

    Steffan JS, Kazantsev A, Spasic-Boskovic O, Greenwald M, Zhu YZ, Gohler H et al (2000) The Huntington’s disease protein interacts with p53 and CREB-binding protein and represses transcription. Proc Natl Acad Sci U S A 97:6763–6768

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  70. 70.

    Shimohata T, Nakajima T, Yamada M, Uchida C, Onodera O, Naruse S et al (2000) Expanded polyglutamine stretches interact with TAFII130, interfering with CREB-dependent transcription. Nat Genet 26:29–36

    CAS  Article  PubMed  Google Scholar 

  71. 71.

    Nguyen H, Jager M, Moretto A, Gruebele M, Kelly JW (2003) Tuning the free-energy landscape of a WW domain by temperature, mutation, and truncation. Proc Natl Acad Sci U S A 100:3948–3953

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  72. 72.

    Ferguson N, Berriman J, Petrovich M, Sharpe TD, Finch JT, Fersht AR (2003) Rapid amyloid fiber formation from the fast-folding WW domain FBP28. Proc Natl Acad Sci U S A 100:9814–9819

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  73. 73.

    Ferguson N, Becker J, Tidow H, Tremmel S, Sharpe TD, Krause G et al (2006) General structural motifs of amyloid protofilaments. Proc Natl Acad Sci U S A 103:16248–16253

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  74. 74.

    Tartaglia GG, Pawar AP, Campioni S, Dobson CM, Chiti F, Vendruscolo M (2008) Prediction of aggregation-prone regions in structured proteins. J Mol Biol 380:425–436

    CAS  Article  PubMed  Google Scholar 

  75. 75.

    Gau SS, Liao HM, Hong CC, Chien WH, Chen CH (2012) Identification of two inherited copy number variants in a male with autism supports two-hit and compound heterozygosity models of autism. Am J Med Genet B Neuropsychiatr Genet 159B:710–717

    Article  PubMed  Google Scholar 

Download references

Acknowledgements

We are grateful to many colleagues for their helpful suggestions, critical discussions, and comments. The technical assistance of Laura Montosa and Eduardo Andrés-León during the confocal microscopy and bioinformatic studies is gratefully acknowledged.

Author information

Affiliations

Authors

Corresponding author

Correspondence to Carlos Suñé.

Ethics declarations

Conflict of Interest

The authors declare that they have no conflict of interest.

Funding

This work was supported by grants from the Spanish Ministry of Economy and Competitiveness (grant number BFU2014–54660-R) and the Andalusian Government (Excellence Project BIO-2515/2012) to C.S. and from the Spanish Ministry of Economy and Competitiveness (grant number BFU2013–44660-R) and the Andalusian Government (Excellence Project CTS-6587) to C.H.M. Support from the European Region Development Fund (ERDF [FEDER]), is also acknowledged.

Electronic supplementary material

ESM 1

(DOCX 3250 kb)

ESM 2

(XLSX 43 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Muñoz-Cobo, J.P., Sánchez-Hernández, N., Gutiérrez, S. et al. Transcriptional Elongation Regulator 1 Affects Transcription and Splicing of Genes Associated with Cellular Morphology and Cytoskeleton Dynamics and Is Required for Neurite Outgrowth in Neuroblastoma Cells and Primary Neuronal Cultures. Mol Neurobiol 54, 7808–7823 (2017). https://doi.org/10.1007/s12035-016-0284-6

Download citation

Keywords

  • TCERG1
  • Transcription
  • Alternative splicing
  • Cytoskeleton
  • Neurite outgrowth
  • Dendrites