The author acknowledges Funda&#231;&#227;o de Amparo &#224; Pesquisa do Estado de S&#227;o Paulo (FAPESP) grant number 2019/21874-5, and Universidade de S&#227;o Paulo for continuous research support.

<ol>
	<li>Wahl, M. C., Will, C. L., L&#252;hrmann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+spliceosome:+design+principles+of+a+dynamic+RNP+machine.">The spliceosome: design principles of a dynamic RNP machine.</a> Cell. 136 (4), 701-718 (2009).</li><li>Scotti, M. M., Swanson, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+mis-splicing+in+disease.">RNA mis-splicing in disease.</a> Nature Reviews Genetics. 17 (1), 19-32 (2016).</li><li>Singh, R. K., Cooper, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pre-mRNA+splicing+in+disease+and+therapeutics.">Pre-mRNA splicing in disease and therapeutics.</a> Trends in Molecular Medicine. 18 (8), 472-482 (2012).</li><li>Staley, J. P., Guthrie, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+devices+of+the+spliceosome:+Motors,+clocks,+springs,+and+things.">Mechanical devices of the spliceosome: Motors, clocks, springs, and things.</a> Cell. 92 (3), 315-326 (1998).</li><li>Wilkinson, M. E., Charenton, C., Nagai, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+splicing+by+the+spliceosome.">RNA splicing by the spliceosome.</a> Annual Review of Biochemistry. 89, 291 (2020).</li><li>Fu, X. D., Ares, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Context-dependent+control+of+alternative+splicing+by+RNA-binding+proteins.">Context-dependent control of alternative splicing by RNA-binding proteins.</a> Nature Reviews Genetics. 15 (10), 689-701 (2014).</li><li>Zhang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+atomic+structure+of+the+human+spliceosome.">An atomic structure of the human spliceosome.</a> Cell. 169 (5), 918-929 (2017).</li><li>Will, C. L., L&#252;hrmann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spliceosome+structure+and+function.">Spliceosome structure and function.</a> Cold Spring Harbor Perspectives in Biology. 3 (7), 003703 (2011).</li><li>Jurica, M. S., Moore, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pre-mRNA+splicing:+Awash+in+a+sea+of+proteins.">Pre-mRNA splicing: Awash in a sea of proteins.</a> Molecular Cell. 12 (1), 5-14 (2003).</li><li>Coltri, P. P., dos Santos, M. G. P., da Silva, G. H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Splicing+and+cancer:+Challenges+and+opportunities.">Splicing and cancer: Challenges and opportunities.</a> Wiley Interdisciplinary Reviews: RNA. 10 (3), 1527 (2019).</li><li>Havens, M. A., Duelli, D. M., Hastings, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+RNA+splicing+for+disease+therapy.">Targeting RNA splicing for disease therapy.</a> Wiley Interdisciplinary Reviews: RNA. 4 (3), 247-266 (2013).</li><li>Pandit, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+analysis+reveals+SR+protein+cooperation+and+competition+in+regulated+splicing.">Genome-wide analysis reveals SR protein cooperation and competition in regulated splicing.</a> Molecular Cell. 50 (2), 223-235 (2013).</li><li>Wang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+complex+network+of+factors+with+overlapping+affinities+represses+splicing+through+intronic+elements.">A complex network of factors with overlapping affinities represses splicing through intronic elements.</a> Nature Structural and Molecular Biology. 20 (1), 36-45 (2013).</li><li>Kornblihtt, A. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+splicing:+A+pivotal+step+between+eukaryotic+transcription+and+translation.">Alternative splicing: A pivotal step between eukaryotic transcription and translation.</a> Nature Reviews Molecular Cell Biology. 14 (3), 153-165 (2013).</li><li>Lee, Y., Rio, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+and+regulation+of+alternative+pre-mRNA+splicing.">Mechanisms and regulation of alternative pre-mRNA splicing.</a> Annual Review of Biochemistry. 84, 291 (2015).</li><li>Dvinge, H., Kim, E., Abdel-Wahab, O., Bradley, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+splicing+factors+as+oncoproteins+and+tumour+suppressors.">RNA splicing factors as oncoproteins and tumour suppressors.</a> Nature Reviews Cancer. 16 (7), 413-430 (2016).</li><li>Vyakarnam, A., Dagher, S. F., Wang, J. L., Patterson, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+a+role+for+galectin-1+in+pre-mRNA+splicing.">Evidence for a role for galectin-1 in pre-mRNA splicing.</a> Molecular and Cellular Biology. 17 (8), 4730-4737 (1997).</li><li>Dagher, S. F., Wang, J. L., Patterson, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+galectin-3+as+a+factor+in+pre-mRNA+splicing.">Identification of galectin-3 as a factor in pre-mRNA splicing.</a> Proceedings of the National Academy of Sciences. 92 (4), 1213-1217 (1995).</li><li>Voss, P. G., Haudek, K. C., Patterson, R. J., Wang, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complementation+of+splicing+activity+by+a+galectin-3-u1+snrnp+complex+on+beads.">Complementation of splicing activity by a galectin-3-u1 snrnp complex on beads.</a> Journal of Visualized Experiments. (166), e61990 (2020).</li><li>Shao, W., Kim, H. -. S., Cao, Y., Xu, Y. -. Z., Query, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+U1-U2+snRNP+interaction+network+during+intron+definition.">A U1-U2 snRNP interaction network during intron definition.</a> Molecular and Cellular Biology. 32 (2), 470-478 (2012).</li><li>Michaels, W. E., Pena-Rasgado, C., Kotaria, R., Bridges, R. J., Hastings, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Open+reading+frame+correction+using+splice-switching+antisense+oligonucleotides+for+the+treatment+of+cystic+fibrosis.">Open reading frame correction using splice-switching antisense oligonucleotides for the treatment of cystic fibrosis.</a> Proceedings of the National Academy of Sciences. 119 (3), 2114886119 (2022).</li><li>Wong, J., Martelly, W., Sharma, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+reporter+based+cellular+assay+for+monitoring+splicing+efficiency.">A reporter based cellular assay for monitoring splicing efficiency.</a> Journal of Visualized Experiments. (175), e63014 (2021).</li><li>Hamid, F. M., Makeyev, E. v. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mechanism+underlying+position-specific+regulation+of+alternative+splicing.">A mechanism underlying position-specific regulation of alternative splicing.</a> Nucleic Acids Research. 45 (21), 12455-12468 (2017).</li><li>vander Feltz, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ACT1-CUP1+assays+determine+the+substrate-specific+sensitivities+of+spliceosomal+mutants+in+budding+yeast.">ACT1-CUP1 assays determine the substrate-specific sensitivities of spliceosomal mutants in budding yeast.</a> Journal of Visualized Experiments. (184), e63232 (2022).</li><li>Plaschka, C., Newman, A. J., Nagai, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+basis+of+nuclear+pre-mRNA+splicing:+Lessons+from+Yeast.">Structural basis of nuclear pre-mRNA splicing: Lessons from Yeast.</a> Cold Spring Harbor Perspectives in Biology. 11 (5), 032391 (2019).</li><li>Stevens, S. W., Abelson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yeast+pre-mRNA+splicing:+methods,+mechanisms,+and+machinery.">Yeast pre-mRNA splicing: methods, mechanisms, and machinery.</a> Methods in Enzymology. 351, 200-220 (2002).</li><li>Lesser, C. F., Guthrie, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutational+analysis+of+pre-mRNA+splicing+in+Saccharomyces+cerevisiae+using+a+sensitive+new+reporter+gene,+CUP1.">Mutational analysis of pre-mRNA splicing in Saccharomyces cerevisiae using a sensitive new reporter gene, CUP1.</a> Genetics. 133 (4), 851-863 (1993).</li><li>Carvalho, F. A., Barros, M. R. A., Girotto, T. P. F., Perona, M. G., Oliveira, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utilization+of+grafix+for+the+detection+of+transient+interactors+of+saccharomyces+cerevisiae+spliceosome+subcomplexes.">Utilization of grafix for the detection of transient interactors of saccharomyces cerevisiae spliceosome subcomplexes.</a> Journal of Visualized Experiments. (165), e61994 (2020).</li><li>Effenberger, K. A., Urabe, V. K., Jurica, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulating+splicing+with+small+molecular+inhibitors+of+the+spliceosome.">Modulating splicing with small molecular inhibitors of the spliceosome.</a> Wiley Interdisciplinary Reviews: RNA. 8 (2), 1381 (2017).</li><li>Corrionero, A., Mi&#241;ana, B., Valc&#225;rcel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduced+fidelity+of+branch+point+recognition+and+alternative+splicing+induced+by+the+anti-tumor+drug+spliceostatin+A.">Reduced fidelity of branch point recognition and alternative splicing induced by the anti-tumor drug spliceostatin A.</a> Genes and Development. 25 (5), 445-459 (2011).</li><li>Basei, F. L., Moura, L. A. R., Kobarg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+the+e1a+minigene+tool+to+study+mrna+splicing+changes.">Using the e1a minigene tool to study mrna splicing changes.</a> Journal of Visualized Experiments. (170), e62181 (2021).</li><li>Volinia, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microRNA+expression+signature+of+human+solid+tumors+defines+cancer+gene+targets.">A microRNA expression signature of human solid tumors defines cancer gene targets.</a> Proceedings of the National Academy of Sciences. 103 (7), 2257-2261 (2006).</li><li>Lu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+expression+profiles+classify+human+cancers.">MicroRNA expression profiles classify human cancers.</a> Nature. 435 (7043), 834-838 (2005).</li><li>Bartel, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metazoan+microRNAs.">Metazoan microRNAs.</a> Cell. 173 (1), 20-51 (2018).</li><li>Gatti da Silva, G. H., Coltri, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+reporter+assay+to+analyze+intronic+microRNA+maturation+in+mammalian+cells.">A reporter assay to analyze intronic microRNA maturation in mammalian cells.</a> Journal of Visualized Experiments. (184), e63498 (2022).</li></ol>

The author has nothing to disclose.

One of the major drivers of cell phenotypes in eukaryotes is gene expression. The control of gene expression is essential and relies on multiple proteins and RNAs, from transcription to translation processes1. Alterations along these steps can trigger several different diseases. RNA processing and splicing constitute an important group of reactions responsible for generating mature RNA molecules2,3,4,5. Splicing removes introns and joins exons to generate a mature RNA molecule capable of performing functional and structural activities. The efficiencies and rates of splicing greatly impact the phenotypes observed under different conditions6.

Splicing is performed by a complex macromolecular machinery named the spliceosome, which is composed of five snRNAs and more than 100 proteins5,7. In addition to core protein components, spliceosomes have trans-acting proteins, which might directly affect the regulation of this process8,9. The importance of splicing is reflected in the broad spectrum of diseases caused by alterations in this process, including neurodegenerative disorders, neuromuscular diseases, and several types of cancer3,10,11. The study of splicing dynamics and spliceosome assembly can greatly impact the understanding of the biology of these disorders.

Spliceosome assembly begins with the association of U1 snRNP with a specific region of the recently transcribed pre-mRNA, the 5&#8217; splice site5,8. The efficient pairing and recognition of 5&#8217; splice sites by U1 snRNA not only define the splice sites that should be used but also control which pre-RNAs will be spliced first, or more efficiently6,12,13. The use of different splice sites can generate multiple alternative isoforms of mature RNAs from the same gene. Alternative splicing is a major source of proteomic diversity, allowing the generation of the RNAs and proteins required during development or in different cells and tissues14,15. Alternative RNA transcripts can generate truncated or non-functional protein isoforms, which might cause diseases10,16. Therefore, the protein components of U1 snRNP complex have an important regulatory role in splicing1. Galectin-1 and Galectin-3 (Gal1 and Gal3) are components of U1 snRNP17,18. Voss et al.19 propose a nuclear extract fractionation method to obtain Gal3-U1 snRNP-enriched complexes capable of rescuing splicing activity in cells depleted of U1. This analysis contributes to understanding the mechanism of Gal3 association to the spliceosomes, and similar approaches can be used to explore other potential splicing regulatory proteins.

The binding of U1 snRNP to 5&#39; splice sites is an important mediator of splicing fidelity20. Mutations at the 5&#8217; splice sites are critical and cause the loss of U1 snRNP pairing, resulting in splicing defects2. Cystic fibrosis, for example, is caused by a mutated 5&#39; splice site in the CFTR gene21. Wong et al.22 describe a method to compensate for 5&#39; splice site mutations with complementary mutations in U1 snRNAs. The approach successfully promotes exon inclusion, generating mature RNAs and resulting in rescue from splicing defects. In addition to offering a possible RNA-based therapy for these diseases, this approach can be useful for the analysis of individual regulatory sequences in pre-mRNAs23.

The fidelity of U1 snRNA association to splice sites is also explored in the yeast model system by van der Feltz24. The budding yeast Saccharomyces cerevisiae has been widely used to explore splicing kinetics and spliceosome assembly25. Splicing kinetics and spliceosome composition are well conserved in eukaryotes, and yeast has been extensively used to monitor the formation of intermediate complexes during assembly26. The ACT1-CUP1 reporter assay is based on the expression of a pair of genes: ACT1, which contains one intron with optimal splice site sequences, and CUP1, which expresses a copper chelator, protecting the cell from the damage caused by this metal ion. CUP1 is only expressed if ACT1 is correctly spliced, therefore creating the reporter system27. Cell growth under conditions with different copper concentrations can be monitored to address the splicing efficiency of different intron sequences or using different regulatory proteins. van der Feltz24 presents a method to analyze the effects of a splicing regulatory protein using this reporter system and copper viability assays. A similar approach can be used to design variations in intron sequences in combination with different proteins to address splicing success in yeasts.

In addition to snRNP association, the binding of proteins is essential during spliceosome assembly1. The control of spliceosome activity is directly dependent on its protein composition and the interactions that occur during its formation. The structural and functional characteristics of protein components are important to activate the complex5,7. Carvalho et al.28 use&#160;an approach based on the Grafix method, which relies on non-reversible protein crosslinking in glycerol gradient analysis. This analysis addresses the composition of proteins and the transient interactions occurring during assembly, which can be critical for the assembly of the active complex and the splicing success. The analysis of other dynamic macromolecular complexes with this approach can be very useful and provide insights into protein organization and interactions.

The chemistry and speed of splicing is also affected by the dynamic association of the different spliceosome components8. Recently, the use of small-molecule splicing inhibitors has revealed that specific steps can be stalled in vitro due to the reduced speed of splicing reactions29,30. Basei et al.31 present a method to analyze alternative splicing on the influence of the splicing regulatory factor, Nek4. The prevalence of transcript isoforms leading to pro- or anti-tumoral phenotypes in HEK293 cells using a minigene is addressed. The misregulation of splicing or different usages of splice sites can lead to alternative transcript generation, causing different types of cancer16. The use of splicing modulators or inhibitors during therapy can determine splice site choices, therefore defining the alternative isoforms generated, whether pro- or anti-tumoral. Paclitaxel and cisplatin are currently used as chemotherapeutic agents. With the use of the E1A minigene, the effects of these drugs in alternative splicing are addressed. Alternative splicing analysis using minigenes is an important strategy to monitor the effects of trans-acting proteins and splicing changes upon therapy and the use of specific drugs.

In this line, many intronic microRNAs (miRNAs) have been reported to be associated with tumoral phenotypes32,33. The control and regulation of intronic miRNA synthesis and biogenesis can be associated with the control of splicing reactions. miRNAs can modulate the stability and prevalence of mRNAs in cells, affecting RNA stability and the rate of translation34 . Gatti da Silva and Coltri35 describe a reporter system to evaluate intronic miRNA biogenesis and maturation, which can be directly linked to alterations in the cellular phenotypes and the determination of pro- or anti-tumoral characteristics. With the modulation of the splicing regulatory protein HuR, the system allows the analysis of the biogenesis and functional activity of an intronic oncogenic miRNA.

The analysis of pre-RNA splicing and the maturation of different RNAs in specific cells will be essential to understand and interpret genomic information. Yeast and mammalian spliceosomes are conserved, and basic research can improve the use of the different methods applied to therapeutics and biomedicine. Despite the large amount of genomic data currently available, the expression of this information in the phenotypes might be cell-specific and dependent on RNA processing and splicing. Understanding the complex regulation of this process will be essential for future research.

pre-rna splicing analysis: from basic science to applied research

ARTICLES DISCUSSED:
Voss, P. G., Haudek, K. C., Patterson, R. J., Wang, J. L. Complementation of splicing activity by a galectin-3-u1 snrnp complex on beads. Journal of Visualized Experiments. (166), e61990 (2020).
Wong, J., Martelly, W., Sharma, S. A reporter based cellular assay for monitoring splicing efficiency. Journal of Visualized Experiments. (175), e63014 (2021).
van der Feltz, C. ACT1-CUP1 assays determine the substrate-specific sensitivities of spliceosomal mutants in budding yeast. Journal of Visualized Experiments. (184), e63232 (2022).
Carvalho, F. A., Barros, M. R. A., Girotto, T. P. F., Perona, M. G., Oliveira, C. C. Utilization of grafix for the detection of transient interactors of saccharomyces cerevisiae spliceosome subcomplexes. Journal of Visualized Experiments. (165), e61994 (2020).
Basei, F. L., Moura, L. A. R., Kobarg, J. Using the E1a minigene tool to study mrna splicing changes. Journal of Visualized Experiments. (170), e62181 (2021).
Gatti da Silva, G. H., Coltri, P. P. A reporter assay to analyze intronic microRNA maturation in mammalian cells. Journal of Visualized Experiments. (184), e63498 (2022).

Read this Scientific Article Protocol about Pre-RNA Splicing Analysis: From Basic Science To Applied Research at JoVE.com

Pre-RNA Splicing Analysis: From Basic Science To Applied Research

ARTICLES DISCUSSED:
Voss, P. G., Haudek, K. C., Patterson, R. J., Wang, J. L. Complementation of splicing activity by a galectin-3-u1 snrnp complex on ...