news and views
An expanding universe of mRNA modifications Samie R Jaffrey
npg
© 2014 Nature America, Inc. All rights reserved.
The fate of an mRNA is regulated by internal base modifications that generate the modified nucleotides N 6-methyladenosine, 5-methylcytosine and inosine. Three new studies show that yeast and human mRNAs also contain pseudouridine residues and that pseudouridylation is induced in various stress states, hinting at a new pathway for post-transcriptional control of mRNA. When we think of mRNA regulation, we usually think of mRNA-binding proteins or microRNAs that get recruited to mRNA in response to cellular signaling events. Emerging evidence suggests that mRNAs can also be regulated by base modifications that are analogous to amino acid modifications in proteins. Three mRNA base modifications have previously been identified, and a fourth type of modified base has now been found in human and yeast mRNA1–3. One of the most well-characterized mRNA base-modification pathways is RNA editing, which involves the conversion of A residues to inosines4. A residues can also be methylated to N6-methyladenosine (m6A)5,6. The presence of m6A was demonstrated with the development of transcriptome-wide m6A-mapping technologies, which showed that m6A residues are present in at least 8,000 transcripts, often near stop codons or within 5′ untranslated regions (UTRs)7,8. These analyses, along with the recent mapping of 5-methylcytosine sites in mammalian mRNA9,10, point to the existence of a diverse landscape of ‘epitranscriptomic’ modifications that can influence mRNA fate and function. The prevalence of mRNA base modifications raises the question of whether there are other base modifications with signaling functions. Pseudouridine is a p articularly compelling candidate because it is found in tRNA, rRNA, small nuclear RNAs (snRNAs) and other noncoding RNAs (ncRNAs), making it the most abundant modified base Samie R. Jaffrey is at the Department of Pharmacology, Weill Cornell Medical College, Cornell University, New York, New York, USA. e-mail: [email protected]
in cells11. Pseudouridine is formed by a remarkable mechanism involving U-base detachment, flipping and reattachment to the ribose12 (Fig. 1a). This isomerization is catalyzed by either the dyskerin pseudouridine synthase (Cbf5 in yeast) or by a family of pseudouridine synthase (Pus) enzymes11. In the case of Cbf5 (dyskerin), pseudouridylation occurs in concert with one of many different small nucleolar RNAs (snoRNAs), which guide the Cbf5 complex to target RNAs and direct pseudouridylation of a central U residue (Fig. 1b). Pus family enzymes, in contrast, modify U residues in specific target sequences, in a snoRNA-independent
a
manner. The idea that mRNA might also be pseudouridylated was originally suggested by the finding that some snoRNAs lack complementarity to ncRNA targets but are complementary to mRNAs13,14. Additionally, there are 23 predicted human pseudouridine synthase genes of unclear function15, thus raising the tantalizing possibility that their endogenous substrates might be mRNAs11. Perhaps most intriguing is the possibility that pseudouridylation can be regulated. Two U residues in the U2 snRNA are individually pseudouridylated by various stress stimuli such as heat shock and nutrient deprivation in yeast 16, and rRNA pseudouridylation
O
O NH
Pseudouridine synthases
180°
N
HN
NH
O
O
Pseudouridine (Ψ)
Uridine
b 5´
5´ NUN Pus1p Pus2p
5´
5´ GUUCNANNC
3´ Cbf5 (dyskerin)
Pus7p 5´
5´ GUΨCNANNC
U 3´
Pus4p 5´
NΨN
UGUAR 3´
3´
snoRNA
5´
UGΨAR
Ψ 3´
Figure 1 Pseudouridylation enzymes that introduce pseudouridine in mRNA. (a) Schematic showing the 180° flip of the U base that is common to the mechanism of all pseudouridine synthases. The wavy line indicates the position of the ribose. (b) RNA-independent and snoRNA-dependent pathways for pseudouridylation contribute to pseudouridine (Ψ) levels in mRNA. Diverse Pus enzymes introduce pseudouridine in mRNA, with Pus7p having a major role in heat shock–induced pseudouridylation. Shown are four Pus family members. Pus1p and Pus2p lack a clear consensus site for pseudouridylation, whereas Pus4p and Pus7p recognize a short sequence motif that directs pseudouridylation. Pseudouridylation mediated by the Cbf5 pathway requires a snoRNA that guides the Cbf5 complex to a target RNA by base complementarity and directs methylation of a central U.
nature structural & molecular biology volume 21 number 11 november 2014
945
npg
© 2014 Nature America, Inc. All rights reserved.
news and views at two sites is regulated by mTOR in mammalian cells17. Three groups now report the first transcriptome-wide maps of pseudouridine, revealing the pseudouridine landscape in yeast and human transcriptomes1–3. Each group used a primer extension–based assay that uses CMC, a chemical that modifies the nitrogen atoms of multiple bases. Whereas most CMC adducts are readily reversed with alkali, CMC-pseudouridine adducts are stable and abort cDNA synthesis by reverse transcriptases18. The position of pseudouridine in various RNAs can thus be determined by mapping the termination sites of their cDNAs18. The three groups used this technique to map pseudouridine in a transcriptomewide manner. This approach, called Psi-seq, Pseudo-seq or Ψ-seq, identified ~50–100 pseudouridine sites in approximately as many mRNAs in yeast grown under nonstress conditions and ~100–400 pseudouridine sites in human cell lines. Pseudouridine was also detected in ncRNAs. The different numbers of pseudouridine sites reported in the different studies relates to the read depth and the different stringency criteria used to call a pseudouridine site. Notably, pseudouridines are not concentrated in specific regions of transcripts but appear to be evenly distributed along 5′ UTRs, coding sequences and 3′ UTRs2,3. One of the most interesting findings is that the levels of pseudouridine in mRNA vary in response to stress states in yeast. Both Lovejoy et al.3 and Schwartz et al.1 found marked increases in the number of pseudouridine sites under heat shock conditions. Schwartz et al.1 found 265 new pseudouridine sites, most of which were formed in a Pus7-dependent manner, similar to the Pus7-dependent pseudouridylation of U2 upon heat shock1,16. Carlile et al.2 found that nutrient stress caused the number of pseudouridine sites to nearly double. The majority of the sites were dependent on Pus1 and Pus7, although other Pus family members also contributed to the pattern of pseudouridylation in these cells2. The overall number of pseudouridine sites in mRNA is considerably less than the number of sites of other modifications such as m6A, but the targeted pseudouridylation of a cohort of transcripts could allow their co-regulation during cellular stress responses. In addition to the action of Pus enzymes, Cbf5-mediated pseudouridylation contributed to the pseudouridine profile, but it did not
appear to influence stress-mediated increases in pseudouridine1. Instead, Cbf5 appears to mediate the smaller number of baseline pseudouridines seen in nonstress conditions. Importantly, not all Cbf5-dependent sites could be linked to a canonical snoRNA1, thus suggesting that other snoRNAs might contribute to mRNA pseudouridylation. An exciting aspect of these studies is their potential disease relevance. X-linked dyskeratosis congenita, mitochondrial myopathy and sideroblastic anemia are each associated with mutations in pseudouridine synthases19,20, and snoRNA42 was recently shown to act as an oncogene in lung cancer21. Profiling pseudouridine might reveal a role for misregulated mRNA pseudouridylation in these diseases. A central question is whether pseudo uridine in mRNA is biologically meaningful. The apparent lack of a dedicated mRNA pseudouridylase raises the possibility that these pseudouridines reflect nonspecific pseudouridylation. For example, a stressinduced upregulation in pseudouridylase activity directed toward rRNA, snRNA and other ncRNAs might modify mRNAs that coincidentally have the same short p seudouridylation-directing motifs. To address this possibility, Lovejoy et al.3 analyzed the sequences surrounding the pseudouridine in RPL11A and TEF1 loci across diverse fungi and found conservation that matched the nearly complete conservation level surrounding the pseudouridine site in the U2 snRNA. A transcriptomewide analysis of the sequence conservation surrounding pseudouridine sites in mRNA could provide further support for functional relevance. Ultimately, a biological role will be revealed by identifying a function for pseudouridine. Because pseudouridine base-pairs with A, and pseudouridine-containing transcripts are translated into functional proteins in living cells22, pseudouridylation does not appear to change the encoded protein sequence. Earlier studies using artificial pseudouridylation showed that pseudouridine at stop codons leads to readthrough23. However, pseudo uridylation of a stop codon was observed in only one endogenous transcript1, thus indicating that promoting readthrough is not its main role. Alternatively, pseudouridine could recruit a yet-unknown pseudouridinebinding protein or influence RNA structure, owing to its altered base-pairing properties11. To explore a role for pseudouridine, Lovejoy et al.3 examined RPL11A and TEF1,
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which are pseudouridylated by Pus1 and Pus4, respectively. They found no change in either the encoded protein sequence or the abundance of mRNA or protein in the Pus-deletion strains3. However, Schwartz et al.1 observed that mRNAs containing heat shock–induced Pus7-dependent pseudouridine sites were 25% more highly expressed in wild-type yeast as compared to Pus7-deficient cells. This raises the possibility that pseudo uridine can stabilize mRNA. Deleting pseudouridine synthases can cause pleiotropic effects due to the loss of pseudouridylation of rRNA, tRNA and other ncRNAs. Thus, the most straightforward approach to determine the role of pseudouridine in mRNA will be to mutate pseudou ridine sites. However, many pseudouridylated mRNAs probably have pseudouridines at a very low stoichiometry, and mutagenesis might not reveal a role for this modification. Thus, identification of the transcripts with the highest pseudouridine stoichiometry and mutagenesis of those U residues will probably be required to reveal the functions of pseudouridine. COMPETING FINANCIAL INTERESTS The author declares no competing financial interests. 1. Schwartz, S. et al. Cell 159, 148–162 (2014). 2. Carlile, T.M. et al. Nature doi:10.1038/nature13802 (5 September 2014). 3. Lovejoy, A.F., Riordan, D.P. & Brown, P.O. PLoS ONE doi:10.1371/journal.pone.0110799 (29 October 2014). 4. Paul, M.S. & Bass, B.L. EMBO J. 17, 1120–1127 (1998). 5. Perry, R.P. & Kelley, D.E. Cell 1, 37–42 (1974). 6. Desrosiers, R., Friderici, K. & Rottman, F. Proc. Natl. Acad. Sci. USA 71, 3971–3975 (1974). 7. Meyer, K.D. et al. Cell 149, 1635–1646 (2012). 8. Dominissini, D. et al. Nature 485, 201–206 (2012). 9. Squires, J.E. et al. Nucleic Acids Res. 40, 5023–5033 (2012). 10. Khoddami, V. & Cairns, B.R. Nat. Biotechnol. 31, 458–464 (2013). 11. Ge, J. & Yu, Y.T. Trends Biochem. Sci. 38, 210–218 (2013). 12. Hoang, C. & Ferre-D’Amare, A.R. Cell 107, 929–939 (2001). 13. Cavaillé, J. et al. Proc. Natl. Acad. Sci. USA 97, 14311–14316 (2000). 14. Hüttenhofer, A., Brosius, J. & Bachellerie, J.P. Curr. Opin. Chem. Biol. 6, 835–843 (2002). 15. Hunter, S. et al. Nucleic Acids Res. 40, D306–D312 (2012). 16. Wu, G., Xiao, M., Yang, C. & Yu, Y.T. EMBO J. 30, 79–89 (2011). 17. Courtes, F.C. et al. J. Biotechnol. 174, 16–21 (2014). 18. Bakin, A. & Ofengand, J. Biochemistry 32, 9754–9762 (1993). 19. Heiss, N.S. et al. Nat. Genet. 19, 32–38 (1998). 20. Bykhovskaya, Y., Casas, K., Mengesha, E., Inbal, A. & Fischel-Ghodsian, N. Am. J. Hum. Genet. 74, 1303–1308 (2004). 21. Mei, Y.P. et al. Oncogene 31, 2794–2804 (2012). 22. Karikó, K. et al. Mol. Ther. 16, 1833–1840 (2008). 23. Karijolich, J. & Yu, Y.T. Nature 474, 395–398 (2011).
An expanding universe of mRNA modifications Samie R Jaffrey
npg
© 2014 Nature America, Inc. All rights reserved.
The fate of an mRNA is regulated by internal base modifications that generate the modified nucleotides N 6-methyladenosine, 5-methylcytosine and inosine. Three new studies show that yeast and human mRNAs also contain pseudouridine residues and that pseudouridylation is induced in various stress states, hinting at a new pathway for post-transcriptional control of mRNA. When we think of mRNA regulation, we usually think of mRNA-binding proteins or microRNAs that get recruited to mRNA in response to cellular signaling events. Emerging evidence suggests that mRNAs can also be regulated by base modifications that are analogous to amino acid modifications in proteins. Three mRNA base modifications have previously been identified, and a fourth type of modified base has now been found in human and yeast mRNA1–3. One of the most well-characterized mRNA base-modification pathways is RNA editing, which involves the conversion of A residues to inosines4. A residues can also be methylated to N6-methyladenosine (m6A)5,6. The presence of m6A was demonstrated with the development of transcriptome-wide m6A-mapping technologies, which showed that m6A residues are present in at least 8,000 transcripts, often near stop codons or within 5′ untranslated regions (UTRs)7,8. These analyses, along with the recent mapping of 5-methylcytosine sites in mammalian mRNA9,10, point to the existence of a diverse landscape of ‘epitranscriptomic’ modifications that can influence mRNA fate and function. The prevalence of mRNA base modifications raises the question of whether there are other base modifications with signaling functions. Pseudouridine is a p articularly compelling candidate because it is found in tRNA, rRNA, small nuclear RNAs (snRNAs) and other noncoding RNAs (ncRNAs), making it the most abundant modified base Samie R. Jaffrey is at the Department of Pharmacology, Weill Cornell Medical College, Cornell University, New York, New York, USA. e-mail: [email protected]
in cells11. Pseudouridine is formed by a remarkable mechanism involving U-base detachment, flipping and reattachment to the ribose12 (Fig. 1a). This isomerization is catalyzed by either the dyskerin pseudouridine synthase (Cbf5 in yeast) or by a family of pseudouridine synthase (Pus) enzymes11. In the case of Cbf5 (dyskerin), pseudouridylation occurs in concert with one of many different small nucleolar RNAs (snoRNAs), which guide the Cbf5 complex to target RNAs and direct pseudouridylation of a central U residue (Fig. 1b). Pus family enzymes, in contrast, modify U residues in specific target sequences, in a snoRNA-independent
a
manner. The idea that mRNA might also be pseudouridylated was originally suggested by the finding that some snoRNAs lack complementarity to ncRNA targets but are complementary to mRNAs13,14. Additionally, there are 23 predicted human pseudouridine synthase genes of unclear function15, thus raising the tantalizing possibility that their endogenous substrates might be mRNAs11. Perhaps most intriguing is the possibility that pseudouridylation can be regulated. Two U residues in the U2 snRNA are individually pseudouridylated by various stress stimuli such as heat shock and nutrient deprivation in yeast 16, and rRNA pseudouridylation
O
O NH
Pseudouridine synthases
180°
N
HN
NH
O
O
Pseudouridine (Ψ)
Uridine
b 5´
5´ NUN Pus1p Pus2p
5´
5´ GUUCNANNC
3´ Cbf5 (dyskerin)
Pus7p 5´
5´ GUΨCNANNC
U 3´
Pus4p 5´
NΨN
UGUAR 3´
3´
snoRNA
5´
UGΨAR
Ψ 3´
Figure 1 Pseudouridylation enzymes that introduce pseudouridine in mRNA. (a) Schematic showing the 180° flip of the U base that is common to the mechanism of all pseudouridine synthases. The wavy line indicates the position of the ribose. (b) RNA-independent and snoRNA-dependent pathways for pseudouridylation contribute to pseudouridine (Ψ) levels in mRNA. Diverse Pus enzymes introduce pseudouridine in mRNA, with Pus7p having a major role in heat shock–induced pseudouridylation. Shown are four Pus family members. Pus1p and Pus2p lack a clear consensus site for pseudouridylation, whereas Pus4p and Pus7p recognize a short sequence motif that directs pseudouridylation. Pseudouridylation mediated by the Cbf5 pathway requires a snoRNA that guides the Cbf5 complex to a target RNA by base complementarity and directs methylation of a central U.
nature structural & molecular biology volume 21 number 11 november 2014
945
npg
© 2014 Nature America, Inc. All rights reserved.
news and views at two sites is regulated by mTOR in mammalian cells17. Three groups now report the first transcriptome-wide maps of pseudouridine, revealing the pseudouridine landscape in yeast and human transcriptomes1–3. Each group used a primer extension–based assay that uses CMC, a chemical that modifies the nitrogen atoms of multiple bases. Whereas most CMC adducts are readily reversed with alkali, CMC-pseudouridine adducts are stable and abort cDNA synthesis by reverse transcriptases18. The position of pseudouridine in various RNAs can thus be determined by mapping the termination sites of their cDNAs18. The three groups used this technique to map pseudouridine in a transcriptomewide manner. This approach, called Psi-seq, Pseudo-seq or Ψ-seq, identified ~50–100 pseudouridine sites in approximately as many mRNAs in yeast grown under nonstress conditions and ~100–400 pseudouridine sites in human cell lines. Pseudouridine was also detected in ncRNAs. The different numbers of pseudouridine sites reported in the different studies relates to the read depth and the different stringency criteria used to call a pseudouridine site. Notably, pseudouridines are not concentrated in specific regions of transcripts but appear to be evenly distributed along 5′ UTRs, coding sequences and 3′ UTRs2,3. One of the most interesting findings is that the levels of pseudouridine in mRNA vary in response to stress states in yeast. Both Lovejoy et al.3 and Schwartz et al.1 found marked increases in the number of pseudouridine sites under heat shock conditions. Schwartz et al.1 found 265 new pseudouridine sites, most of which were formed in a Pus7-dependent manner, similar to the Pus7-dependent pseudouridylation of U2 upon heat shock1,16. Carlile et al.2 found that nutrient stress caused the number of pseudouridine sites to nearly double. The majority of the sites were dependent on Pus1 and Pus7, although other Pus family members also contributed to the pattern of pseudouridylation in these cells2. The overall number of pseudouridine sites in mRNA is considerably less than the number of sites of other modifications such as m6A, but the targeted pseudouridylation of a cohort of transcripts could allow their co-regulation during cellular stress responses. In addition to the action of Pus enzymes, Cbf5-mediated pseudouridylation contributed to the pseudouridine profile, but it did not
appear to influence stress-mediated increases in pseudouridine1. Instead, Cbf5 appears to mediate the smaller number of baseline pseudouridines seen in nonstress conditions. Importantly, not all Cbf5-dependent sites could be linked to a canonical snoRNA1, thus suggesting that other snoRNAs might contribute to mRNA pseudouridylation. An exciting aspect of these studies is their potential disease relevance. X-linked dyskeratosis congenita, mitochondrial myopathy and sideroblastic anemia are each associated with mutations in pseudouridine synthases19,20, and snoRNA42 was recently shown to act as an oncogene in lung cancer21. Profiling pseudouridine might reveal a role for misregulated mRNA pseudouridylation in these diseases. A central question is whether pseudo uridine in mRNA is biologically meaningful. The apparent lack of a dedicated mRNA pseudouridylase raises the possibility that these pseudouridines reflect nonspecific pseudouridylation. For example, a stressinduced upregulation in pseudouridylase activity directed toward rRNA, snRNA and other ncRNAs might modify mRNAs that coincidentally have the same short p seudouridylation-directing motifs. To address this possibility, Lovejoy et al.3 analyzed the sequences surrounding the pseudouridine in RPL11A and TEF1 loci across diverse fungi and found conservation that matched the nearly complete conservation level surrounding the pseudouridine site in the U2 snRNA. A transcriptomewide analysis of the sequence conservation surrounding pseudouridine sites in mRNA could provide further support for functional relevance. Ultimately, a biological role will be revealed by identifying a function for pseudouridine. Because pseudouridine base-pairs with A, and pseudouridine-containing transcripts are translated into functional proteins in living cells22, pseudouridylation does not appear to change the encoded protein sequence. Earlier studies using artificial pseudouridylation showed that pseudouridine at stop codons leads to readthrough23. However, pseudo uridylation of a stop codon was observed in only one endogenous transcript1, thus indicating that promoting readthrough is not its main role. Alternatively, pseudouridine could recruit a yet-unknown pseudouridinebinding protein or influence RNA structure, owing to its altered base-pairing properties11. To explore a role for pseudouridine, Lovejoy et al.3 examined RPL11A and TEF1,
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which are pseudouridylated by Pus1 and Pus4, respectively. They found no change in either the encoded protein sequence or the abundance of mRNA or protein in the Pus-deletion strains3. However, Schwartz et al.1 observed that mRNAs containing heat shock–induced Pus7-dependent pseudouridine sites were 25% more highly expressed in wild-type yeast as compared to Pus7-deficient cells. This raises the possibility that pseudo uridine can stabilize mRNA. Deleting pseudouridine synthases can cause pleiotropic effects due to the loss of pseudouridylation of rRNA, tRNA and other ncRNAs. Thus, the most straightforward approach to determine the role of pseudouridine in mRNA will be to mutate pseudou ridine sites. However, many pseudouridylated mRNAs probably have pseudouridines at a very low stoichiometry, and mutagenesis might not reveal a role for this modification. Thus, identification of the transcripts with the highest pseudouridine stoichiometry and mutagenesis of those U residues will probably be required to reveal the functions of pseudouridine. COMPETING FINANCIAL INTERESTS The author declares no competing financial interests. 1. Schwartz, S. et al. Cell 159, 148–162 (2014). 2. Carlile, T.M. et al. Nature doi:10.1038/nature13802 (5 September 2014). 3. Lovejoy, A.F., Riordan, D.P. & Brown, P.O. PLoS ONE doi:10.1371/journal.pone.0110799 (29 October 2014). 4. Paul, M.S. & Bass, B.L. EMBO J. 17, 1120–1127 (1998). 5. Perry, R.P. & Kelley, D.E. Cell 1, 37–42 (1974). 6. Desrosiers, R., Friderici, K. & Rottman, F. Proc. Natl. Acad. Sci. USA 71, 3971–3975 (1974). 7. Meyer, K.D. et al. Cell 149, 1635–1646 (2012). 8. Dominissini, D. et al. Nature 485, 201–206 (2012). 9. Squires, J.E. et al. Nucleic Acids Res. 40, 5023–5033 (2012). 10. Khoddami, V. & Cairns, B.R. Nat. Biotechnol. 31, 458–464 (2013). 11. Ge, J. & Yu, Y.T. Trends Biochem. Sci. 38, 210–218 (2013). 12. Hoang, C. & Ferre-D’Amare, A.R. Cell 107, 929–939 (2001). 13. Cavaillé, J. et al. Proc. Natl. Acad. Sci. USA 97, 14311–14316 (2000). 14. Hüttenhofer, A., Brosius, J. & Bachellerie, J.P. Curr. Opin. Chem. Biol. 6, 835–843 (2002). 15. Hunter, S. et al. Nucleic Acids Res. 40, D306–D312 (2012). 16. Wu, G., Xiao, M., Yang, C. & Yu, Y.T. EMBO J. 30, 79–89 (2011). 17. Courtes, F.C. et al. J. Biotechnol. 174, 16–21 (2014). 18. Bakin, A. & Ofengand, J. Biochemistry 32, 9754–9762 (1993). 19. Heiss, N.S. et al. Nat. Genet. 19, 32–38 (1998). 20. Bykhovskaya, Y., Casas, K., Mengesha, E., Inbal, A. & Fischel-Ghodsian, N. Am. J. Hum. Genet. 74, 1303–1308 (2004). 21. Mei, Y.P. et al. Oncogene 31, 2794–2804 (2012). 22. Karikó, K. et al. Mol. Ther. 16, 1833–1840 (2008). 23. Karijolich, J. & Yu, Y.T. Nature 474, 395–398 (2011).
