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Craving for Introns
Molecular Cell
Spotlight Craving for Introns Michela Zaffagni1 and Sebastian Kadener1,* 1Department of Biology, Brandeis University, 415 South Street, Waltham, MA 02453, USA *Correspondence: [email protected] https://doi.org/10.1016/j.molcel.2019.03.008
Parenteau et al. (2019) and Morgan et al. (2019) showed that a subset of introns can work as non-coding RNAs that trap the spliceosome and decrease global splicing upon nutrient depletion in yeast, providing a new example of the functionality of introns, molecules that were previously assumed to be useless. One of the most important challenges of molecular biology is determining the functionality of genomic elements. Biochemical and genetic experiments, as well as evolutionary conservation, play a pivotal role in understanding if and how DNA and RNA sequences function. Theoretically and practically, this is not simple, as in many cases these sequences are important only in certain situations that can be difficult to imagine a priori. In two back-to-back articles recently published in Nature, Parenteau et al. (2019) and Morgan et al. (2019) describe a new class of spliceosome-sequestering introns which play a key role upon nutrient deprivation. As almost all introns are considered to be a non-functional sub-product of RNA splicing, this was a surprising and exciting result. Pre-mRNA splicing is the process by which introns are removed from a primary transcript, permitting exons to be joined together to produce a mature mRNA. Although introns contain sequences that modulate the initial steps of splicing, these segments of RNA were thought to be immediately degraded once excised (Figure 1A). When introns are retained in mRNAs, they can mediate decay (Lewis et al., 2003) or localization (Buckley et al., 2011). Furthermore, some small nucleolar RNAs, microRNAs, and long non-coding RNAs are embedded within introns. However, these examples of functional introns seem to be the exceptions that confirm the rule. Parenteau et al. (2019) and Morgan et al. (2019) overturn this concept by showing that a variety of retained and excised introns can broadly modulate gene expression. Both groups chose the perfect system for their analyses; S. cerevisiae has only 289 genes that contain introns, making it suitable for systematic intron deletion
studies. With that in mind, Parenteau et al. (2019) created a complete collection of yeast strains in which individual introns were deleted. Many of these strains showed reduced survival upon nutrient depletion. Importantly, growth was not restored by the expression of the hostgene cDNAs or by expression of the introns themselves. By contrast, introduction of constructs carrying mutations that block intron excision but allow the association with splicing factors and that include the 50 untranslated regions (50 UTR) of the hosting mRNA rescue the intron-depletion phenotype. Intriguingly, the genes hosting these introns encode proteins with diverse functions. RNA sequencing experiments showed that under starvation conditions there was a global alteration of splicing in many intron-deletion strains. As a result, the expression of genes encoding proteins involved in translation and respiration was particularly reduced. From these data and RNA structure prediction analysis, Parenteau et al. concluded that, during nutrient depletion, these introns form hairpins with the 50 UTRs of the mRNAs. The authors propose that these exonintron structures sequester spliceosomal proteins and alter global splicing. In this way, these introns indirectly disrupt ribosome synthesis, which results in an energy savings during starvation. However, how the hairpin is formed and how it mediates splicing regulation remain unclear. Using another experimental approach, Morgan et al. (2019) also concluded that certain yeast introns function as ‘‘spliceosome traps’’ during starvation. Their study is based on RNA sequencing data that showed a strong accumulation of 34 spliced introns in yeast cells from saturated cultures. Intriguingly, these introns do not accumulate as lariats or circular
structures but as linear molecules, suggesting that their stabilization occurs late (or after) the splicing cycle. Pull-down experiments proved that there is a direct association between these ‘‘linear introns’’ and the intron-lariat-spliceosome complex during the stationary phase. Furthermore, the excised introns are stabilized upon extended inhibition of TORC1, a master regulator of cell growth, and by secretory stress induced by tunicamycin and dithiothreitol treatment, suggesting that different types of stress trigger a similar growth-repressing mechanism. These data indicate that under stress, some released introns sequester specific spliceosome components and modulate splicing. In sum, these studies define two classes of spliceosome-sequestering introns. The introns described in Parenteau et al. (2019) are retained and form a hairpin with the 50 UTR, whereas the introns reported in Morgan et al. (2019) are excised from the pre-mRNAs and stabilized by direct interaction with spliceosomal proteins. Notably, only two genes host an intron that belongs to both categories, implying that the two pathways are likely mutually exclusive. Both models propose that some introns work as decoy molecules that sequester spliceosomal proteins and diminish the output of highly expressed genes, such as ribosomal proteins and metabolic enzymes, which saves energy during starvation (Figure 1B). Intriguingly, neither group was able to identify any consensus sequence or suggest that the recognition is based on the RNA structure. However, it is unclear how the starvation signal affects only specific introns and how the system returns to a normal state after stress removal. For instance, Parentau et al. show that 94% of the intron-deleted
Molecular Cell 73, March 21, 2019 ª 2019 Elsevier Inc. 1095
Molecular Cell
Spotlight
A
B
snRNA U1 binding to nascent RNA increases the elongation rate of RNA polymerase II (Damgaard et al., 2008) and also protects pre-mRNAs from premature cleavage (Kaida et al., 2010). In addition, intron-exon circular RNAs bind to U1 and RNA polymerase II and enhance transcription of the mRNAs from their hosting genes (Li et al., 2015). Although the molecular details of intron-mediated inhibition of splicing remain to be elucidated, it is now clear that intronic regions of RNA transcripts, in most cases assumed to have no functionality, are active participants in cellular function. REFERENCES Boutz, P.L., Bhutkar, A., and Sharp, P.A. (2015). Detained introns are a novel, widespread class of post-transcriptionally spliced introns. Genes Dev. 29, 63–80. Buckley, P.T., Lee, M.T., Sul, J., Miyashiro, K.Y., Bell, T.J., and Fisher, S.A. (2011). Cytoplasmic intron sequence-retaining transcripts can be dendritically targeted via ID element retrotransposons. Neuron 69, 877–884. Damgaard, C.K., Kahns, S., Lykke-Andersen, S., Nielsen, A.L., Jensen, T.H., and Kjems, J. (2008). A 50 splice site enhances the recruitment of basal transcription initiation factors in vivo. Mol. Cell 29, 271–278.
Figure 1. Craving for Introns (A) During the log phase, introns are removed from the pre-mRNAs and degraded. The spliceosome can be recycled, and yeast growth is supported by high protein synthesis. (B) During the stationary phase, some introns are stabilized by binding to spliceosomal proteins. These introns can be either retained in the mRNA and form a hairpin structure with their close exon or excised and remain in a linear form. As a result, the spliceosome is sequestered, and pre-mRNAs of highly expressed genes, such as ribosomal proteins (RPGs), accumulate. Finally, gene expression is altered, ribosomes are not assembled, and the cell can save energy for overcoming starvation.
strains showed a growth phenotype upon nutrient depletion; however, only few of the introns targeted in the growth-deficient strains can form the abovementioned hairpin structure. Furthermore, it will be interesting to know whether other stresses in addition to starvation activate the process. One intriguing possibility is that mechanism could help cells to integrate mild stresses over time, each of which slightly alter the stability of those introns. Does a similar stress response mechanism exist in other eukaryotes? Indeed,
1096 Molecular Cell 73, March 21, 2019
global inhibition of splicing is used by mammalian cells to diminish the expression of non-stress-relevant genes during heat shock (Shalgi et al., 2014) and upon DNA damage (Boutz et al., 2015). Taken together, these reports suggest that global splicing repression is a conserved stress-response mechanism. However, the significantly larger number of introns in metazoans suggests that it is unlikely that introns can modulate global splicing by working in trans. Nevertheless, studies in metazoans suggest that a comparable process may act in cis. For example,
Kaida, D., Berg, M.G., Younis, I., Kasim, M., Singh, L.N., Wan, L., and Dreyfuss, G. (2010). U1 snRNP protects pre-mRNAs from premature cleavage and polyadenylation. Nature 468, 664–668. Lewis, B.P., Green, R.E., and Brenner, S.E. (2003). Evidence for the widespread coupling of alternative splicing and nonsense-mediated mRNA decay in humans. Proc. Natl. Acad. Sci. USA 100, 189–192. Li, Z., Huang, C., Bao, C., Chen, L., Lin, M., Wang, X., Zhong, G., Yu, B., Hu, W., Dai, L., et al. (2015). Exon-intron circular RNAs regulate transcription in the nucleus. Nat. Struct. Mol. Biol. 22, 256–264. Morgan, J.T., Fink, G.R., and Bartel, D.P. (2019). Excised linear introns regulate growth in yeast. Nature 565, 606–611. Parenteau, J., Maignon, L., Berthoumieux, M., Catala, M., Gagnon, V., and Abou Elela, S. (2019). Introns are mediators of cell response to starvation. Nature 565, 612–617. Shalgi, R., Hurt, J.A., Lindquist, S., and Burge, C.B. (2014). Widespread inhibition of posttranscriptional splicing shapes the cellular transcriptome following heat shock. Cell Rep. 7, 1362–1370.
Spotlight Craving for Introns Michela Zaffagni1 and Sebastian Kadener1,* 1Department of Biology, Brandeis University, 415 South Street, Waltham, MA 02453, USA *Correspondence: [email protected] https://doi.org/10.1016/j.molcel.2019.03.008
Parenteau et al. (2019) and Morgan et al. (2019) showed that a subset of introns can work as non-coding RNAs that trap the spliceosome and decrease global splicing upon nutrient depletion in yeast, providing a new example of the functionality of introns, molecules that were previously assumed to be useless. One of the most important challenges of molecular biology is determining the functionality of genomic elements. Biochemical and genetic experiments, as well as evolutionary conservation, play a pivotal role in understanding if and how DNA and RNA sequences function. Theoretically and practically, this is not simple, as in many cases these sequences are important only in certain situations that can be difficult to imagine a priori. In two back-to-back articles recently published in Nature, Parenteau et al. (2019) and Morgan et al. (2019) describe a new class of spliceosome-sequestering introns which play a key role upon nutrient deprivation. As almost all introns are considered to be a non-functional sub-product of RNA splicing, this was a surprising and exciting result. Pre-mRNA splicing is the process by which introns are removed from a primary transcript, permitting exons to be joined together to produce a mature mRNA. Although introns contain sequences that modulate the initial steps of splicing, these segments of RNA were thought to be immediately degraded once excised (Figure 1A). When introns are retained in mRNAs, they can mediate decay (Lewis et al., 2003) or localization (Buckley et al., 2011). Furthermore, some small nucleolar RNAs, microRNAs, and long non-coding RNAs are embedded within introns. However, these examples of functional introns seem to be the exceptions that confirm the rule. Parenteau et al. (2019) and Morgan et al. (2019) overturn this concept by showing that a variety of retained and excised introns can broadly modulate gene expression. Both groups chose the perfect system for their analyses; S. cerevisiae has only 289 genes that contain introns, making it suitable for systematic intron deletion
studies. With that in mind, Parenteau et al. (2019) created a complete collection of yeast strains in which individual introns were deleted. Many of these strains showed reduced survival upon nutrient depletion. Importantly, growth was not restored by the expression of the hostgene cDNAs or by expression of the introns themselves. By contrast, introduction of constructs carrying mutations that block intron excision but allow the association with splicing factors and that include the 50 untranslated regions (50 UTR) of the hosting mRNA rescue the intron-depletion phenotype. Intriguingly, the genes hosting these introns encode proteins with diverse functions. RNA sequencing experiments showed that under starvation conditions there was a global alteration of splicing in many intron-deletion strains. As a result, the expression of genes encoding proteins involved in translation and respiration was particularly reduced. From these data and RNA structure prediction analysis, Parenteau et al. concluded that, during nutrient depletion, these introns form hairpins with the 50 UTRs of the mRNAs. The authors propose that these exonintron structures sequester spliceosomal proteins and alter global splicing. In this way, these introns indirectly disrupt ribosome synthesis, which results in an energy savings during starvation. However, how the hairpin is formed and how it mediates splicing regulation remain unclear. Using another experimental approach, Morgan et al. (2019) also concluded that certain yeast introns function as ‘‘spliceosome traps’’ during starvation. Their study is based on RNA sequencing data that showed a strong accumulation of 34 spliced introns in yeast cells from saturated cultures. Intriguingly, these introns do not accumulate as lariats or circular
structures but as linear molecules, suggesting that their stabilization occurs late (or after) the splicing cycle. Pull-down experiments proved that there is a direct association between these ‘‘linear introns’’ and the intron-lariat-spliceosome complex during the stationary phase. Furthermore, the excised introns are stabilized upon extended inhibition of TORC1, a master regulator of cell growth, and by secretory stress induced by tunicamycin and dithiothreitol treatment, suggesting that different types of stress trigger a similar growth-repressing mechanism. These data indicate that under stress, some released introns sequester specific spliceosome components and modulate splicing. In sum, these studies define two classes of spliceosome-sequestering introns. The introns described in Parenteau et al. (2019) are retained and form a hairpin with the 50 UTR, whereas the introns reported in Morgan et al. (2019) are excised from the pre-mRNAs and stabilized by direct interaction with spliceosomal proteins. Notably, only two genes host an intron that belongs to both categories, implying that the two pathways are likely mutually exclusive. Both models propose that some introns work as decoy molecules that sequester spliceosomal proteins and diminish the output of highly expressed genes, such as ribosomal proteins and metabolic enzymes, which saves energy during starvation (Figure 1B). Intriguingly, neither group was able to identify any consensus sequence or suggest that the recognition is based on the RNA structure. However, it is unclear how the starvation signal affects only specific introns and how the system returns to a normal state after stress removal. For instance, Parentau et al. show that 94% of the intron-deleted
Molecular Cell 73, March 21, 2019 ª 2019 Elsevier Inc. 1095
Molecular Cell
Spotlight
A
B
snRNA U1 binding to nascent RNA increases the elongation rate of RNA polymerase II (Damgaard et al., 2008) and also protects pre-mRNAs from premature cleavage (Kaida et al., 2010). In addition, intron-exon circular RNAs bind to U1 and RNA polymerase II and enhance transcription of the mRNAs from their hosting genes (Li et al., 2015). Although the molecular details of intron-mediated inhibition of splicing remain to be elucidated, it is now clear that intronic regions of RNA transcripts, in most cases assumed to have no functionality, are active participants in cellular function. REFERENCES Boutz, P.L., Bhutkar, A., and Sharp, P.A. (2015). Detained introns are a novel, widespread class of post-transcriptionally spliced introns. Genes Dev. 29, 63–80. Buckley, P.T., Lee, M.T., Sul, J., Miyashiro, K.Y., Bell, T.J., and Fisher, S.A. (2011). Cytoplasmic intron sequence-retaining transcripts can be dendritically targeted via ID element retrotransposons. Neuron 69, 877–884. Damgaard, C.K., Kahns, S., Lykke-Andersen, S., Nielsen, A.L., Jensen, T.H., and Kjems, J. (2008). A 50 splice site enhances the recruitment of basal transcription initiation factors in vivo. Mol. Cell 29, 271–278.
Figure 1. Craving for Introns (A) During the log phase, introns are removed from the pre-mRNAs and degraded. The spliceosome can be recycled, and yeast growth is supported by high protein synthesis. (B) During the stationary phase, some introns are stabilized by binding to spliceosomal proteins. These introns can be either retained in the mRNA and form a hairpin structure with their close exon or excised and remain in a linear form. As a result, the spliceosome is sequestered, and pre-mRNAs of highly expressed genes, such as ribosomal proteins (RPGs), accumulate. Finally, gene expression is altered, ribosomes are not assembled, and the cell can save energy for overcoming starvation.
strains showed a growth phenotype upon nutrient depletion; however, only few of the introns targeted in the growth-deficient strains can form the abovementioned hairpin structure. Furthermore, it will be interesting to know whether other stresses in addition to starvation activate the process. One intriguing possibility is that mechanism could help cells to integrate mild stresses over time, each of which slightly alter the stability of those introns. Does a similar stress response mechanism exist in other eukaryotes? Indeed,
1096 Molecular Cell 73, March 21, 2019
global inhibition of splicing is used by mammalian cells to diminish the expression of non-stress-relevant genes during heat shock (Shalgi et al., 2014) and upon DNA damage (Boutz et al., 2015). Taken together, these reports suggest that global splicing repression is a conserved stress-response mechanism. However, the significantly larger number of introns in metazoans suggests that it is unlikely that introns can modulate global splicing by working in trans. Nevertheless, studies in metazoans suggest that a comparable process may act in cis. For example,
Kaida, D., Berg, M.G., Younis, I., Kasim, M., Singh, L.N., Wan, L., and Dreyfuss, G. (2010). U1 snRNP protects pre-mRNAs from premature cleavage and polyadenylation. Nature 468, 664–668. Lewis, B.P., Green, R.E., and Brenner, S.E. (2003). Evidence for the widespread coupling of alternative splicing and nonsense-mediated mRNA decay in humans. Proc. Natl. Acad. Sci. USA 100, 189–192. Li, Z., Huang, C., Bao, C., Chen, L., Lin, M., Wang, X., Zhong, G., Yu, B., Hu, W., Dai, L., et al. (2015). Exon-intron circular RNAs regulate transcription in the nucleus. Nat. Struct. Mol. Biol. 22, 256–264. Morgan, J.T., Fink, G.R., and Bartel, D.P. (2019). Excised linear introns regulate growth in yeast. Nature 565, 606–611. Parenteau, J., Maignon, L., Berthoumieux, M., Catala, M., Gagnon, V., and Abou Elela, S. (2019). Introns are mediators of cell response to starvation. Nature 565, 612–617. Shalgi, R., Hurt, J.A., Lindquist, S., and Burge, C.B. (2014). Widespread inhibition of posttranscriptional splicing shapes the cellular transcriptome following heat shock. Cell Rep. 7, 1362–1370.