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Viroids & the RNA World: from genomic scale (RNA) to atomic scale (ribozyme)
Fabrice Leclerc
To cite this version:
Fabrice Leclerc. Viroids & the RNA World: from genomic scale (RNA) to atomic scale (ribozyme).
Master. BGA Biochimie et Génétique des ARN, Paris, France. 2020. �hal-03313809�
Viroids
&
the RNA World
G G
A A G
A G
A U
U G A A G
A C G A G U G A A C A U U A U U U U
U U A A U
A A A A
G U
U C
A C C
A C G
A C U C C U C
C U U C U C U
C A C A A
G U C G
AAA C U C A
G A G U
C
G G A A A G U C
G G A AC A
G A C C U G G U U U C
G U C A A A
C A A A
G U U U A A
U C A
U A
U C C U C A
C U U C U U G U U C
U A A U A
A
A C A A G A U U U U G U A A A A A A A A C UA G A A AG AU G A G G A A U A A A C C U U G G C A A G C U C A U C A UG G U C U U U C C A C U C U U C U C C G U A A G A A G C A G A G G U U A C 1
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1
Fabrice Leclerc
Institute for Integrative Biology of the Cell (I2BC)
CNRS-CEA-Univ. Paris Saclay
fabrice.leclerc@i2bc.paris-saclay.fr
Viroids
&
the RNA World
from genomic scale (RNA) to
atomic scale (ribozyme)
Viroid (ASBVd)
G G
A A G
A G
A U
U G A A G
A C G A G U G A A C A U U A U U U U
U U A A U
A A A A
G U
U C
A C C
A C G
A C U C C U C
C U U C U C U
C A C A A
G U C G
AAA C U C A
G A G U
C
G G A A A G U C
G G A AC A
G A C C U G G U U U C
G U C A A A
C A A A
G U U U A A
U C A
U A
U C C U C A
C U U C U U G U U C
U A A U A
A
A C A A G A U U U U G U A A A A A A A A C UA G A A AG AU G A G G A A U A A A C C U U G G C A A G C U C A U C A UG G U C U U U C C A C U C U U C U C C G U A A G A A G C A G A G G U U A C 1
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79-nt
249-nt
HHRz
2
Introduction
• evolution (RNA world)& genomics
• RNA biology & RNomics (molecular and cellular functions)
• structural biology & structural
bioinformatics (structural basis for functions)
• enzymology & computational enzymology (catalysis)
3
Prebiotic & RNA Worlds
selfish elements, viroids,
etc self-replication replication,
amplification
Horning & Joyce, PNAS, 2016.
Attwater et al., Nat. Chem., 2013.
ribozymes
Martin et al., Life, 2015.
4
Virus World
&
RNA World
« The ancient Virus World and evolution of cells »
Biology Direct 2006, 1:29 http://www.biology-direct.com/content/1/1/29
Page 12 of 27
(page number not for citation purposes)
Evolution of the virus world: origin of the main lineages from the primordial gene pool Figure 2
Evolution of the virus world: origin of the main lineages from the primordial gene pool. Characteristic images of RNA and protein structures are shown for each postulated stage of evolution, and characteristic virion images are shown for the emerging classes of viruses. Thin arrows show the postulated movement of genetic pools between inorganic compart- ments. Block arrows show the origin of different classes of viruses at different stages of pre-cellular evolution.
Pre-archaeal
compartment Pre-bacterial compartment
ocean
selfish ribozymes (group I Introns) positive-
strand, ds RNA viruses retrons, group II introns
crust
dsDNA and RCR viruses, plasmids Bacteria with
plasmids, retrons, group I & II introns Archaea with
plasmids, group I introns
Escape of cells with their viruses and other
parasitic elements
RNA World RNA-DNA Retro
World
inorganic compartments RNA-Protein World
DNA World
Koonin et al., Biol. Direct, 2006
viroids
5
Viroids: « survivors from the RNA World »
• small size
• high GC
• circRNA
• periodicity
• no protein- coding
• ribozyme
• error-prone replication
• replication fidelity
• replication
• genome
assembly
• ribosome-
free
• replication
features
functions
Holmes, J. Virol., 2011
6
Viroids: Families
Vol. 20, No. 1, 2007 / 11 an in vitro transcription system. With a combination of proto-
cols to remove cellular RNAs and, thereby, enrich the de novo synthesized (–)-strand PSTVd RNAs from the circular (+)- RNA templates in potato nuclear extracts and primer extension, Kolonko and associates (2006) mapped the transcription initiate site on the circular (+)-RNA to U359/C1 of the left terminal loop (Fig. 1). Because of the low resolution of sequencing gels, it is not possible to determine precisely whether U359 or C1 is the exact initiation site. Site-directed mutagenesis in combination with infection studies in tomato revealed that the C1G mutation was maintained stably, whereas U359G reverted to wild type, suggesting that perhaps U359 is the bona fide initiation site. It is notable that these data are consistent with previous in vitro studies showing that Pol II binds to the terminal loop or loops of PSTVd (Goodman et al. 1984). These observations establish a basis for further investigations to determine whether the in vitro transcription initiation site is the same as that used in vivo.
The transcription initiation sites on the (–)-strand template also remain to be determined.
A critical issue for all viroids is that bona fide promoter se- quences have not been characterized fully. This obviously is one of the most pressing issues that need to be addressed in order to fully understand how viroid RNA templates are recognized and transcribed by the cellular machinery.
RNA motifs and protein factors for cleavage and ligation.
The sequence and structural conservation of the CCR of several members of Pospiviroidae suggests its potential importance in
viroid processing during replication (Candresse et al. 1990;
Diener 1986; Hashimoto and Machida 1985; Meshi et al.
1985; Tabler and Sänger 1985; Visvader et al. 1985). Extensive in vitro studies provided evidence to support this hypothesis (Baumstark and Riesner 1995). Furthermore, in vitro studies with longer than unit-length PSTVd transcripts mapped the cleavage and ligation site to between G95 and G96 (Baumstark et al. 1997). The first cleavage at the 5′ end of G96 occurs in a metastable tetraloop motif, which results in a conformational change to form a stable loop E that drives the second cleavage at the 3′ end of G95 and subsequent ligation (Baumstark et al.
1997). Recent work with a minicircle RNA showed that the CCR contains all the necessary elements for cleavage and liga- tion (Schrader et al. 2003). It is important to note that process- ing also can occur outside CCR, with the specific sites to be elucidated (Hammond et al. 1989; Tabler et al. 1992). A key question that remains to be answered is whether single or mul- tiple sites are used for processing in vivo.
Weak self-cleavage of PSTVd RNAs has been reported by some researchers (Robertson et al. 1985) but not by others (Tabler and Sänger 1985; Tsagris et al. 1987a,b). It generally is thought that a cellular RNase which remains to be identified catalyzes the cleavage of concatemeric RNAs (Tsagris et al.
1987a,b). Reasoning that the general difficulty of demonstrat- ing self-cleavage of RNAs in Pospiviroidae could be attributed to the interference of nonribozyme RNA sequences in the sub- strates used during in vitro assays, Liu and Symons (1998)
Fig. 3. Asymmetric rolling circle replication of Potato spindle tuber viroid (PSTVd) and symmetric rolling circle replication of Avocado sunblotch viroid (ASBVd). The secondary structures of the genomic or circular RNAs are sketched to facilitate illustration of the approximate transcription initiation sites.
Vol. 20, No. 1, 2007 / 11 an in vitro transcription system. With a combination of proto-
cols to remove cellular RNAs and, thereby, enrich the de novo synthesized (–)-strand PSTVd RNAs from the circular (+)- RNA templates in potato nuclear extracts and primer extension, Kolonko and associates (2006) mapped the transcription initiate site on the circular (+)-RNA to U359/C1 of the left terminal loop (Fig. 1). Because of the low resolution of sequencing gels, it is not possible to determine precisely whether U359 or C1 is the exact initiation site. Site-directed mutagenesis in combination with infection studies in tomato revealed that the C1G mutation was maintained stably, whereas U359G reverted to wild type, suggesting that perhaps U359 is the bona fide initiation site. It is notable that these data are consistent with previous in vitro studies showing that Pol II binds to the terminal loop or loops of PSTVd (Goodman et al. 1984). These observations establish a basis for further investigations to determine whether the in vitro transcription initiation site is the same as that used in vivo.
The transcription initiation sites on the (–)-strand template also remain to be determined.
A critical issue for all viroids is that bona fide promoter se- quences have not been characterized fully. This obviously is one of the most pressing issues that need to be addressed in order to fully understand how viroid RNA templates are recognized and transcribed by the cellular machinery.
RNA motifs and protein factors for cleavage and ligation.
The sequence and structural conservation of the CCR of several members of Pospiviroidae suggests its potential importance in
viroid processing during replication (Candresse et al. 1990;
Diener 1986; Hashimoto and Machida 1985; Meshi et al.
1985; Tabler and Sänger 1985; Visvader et al. 1985). Extensive in vitro studies provided evidence to support this hypothesis (Baumstark and Riesner 1995). Furthermore, in vitro studies with longer than unit-length PSTVd transcripts mapped the cleavage and ligation site to between G95 and G96 (Baumstark et al. 1997). The first cleavage at the 5′ end of G96 occurs in a metastable tetraloop motif, which results in a conformational change to form a stable loop E that drives the second cleavage at the 3′ end of G95 and subsequent ligation (Baumstark et al.
1997). Recent work with a minicircle RNA showed that the CCR contains all the necessary elements for cleavage and liga- tion (Schrader et al. 2003). It is important to note that process- ing also can occur outside CCR, with the specific sites to be elucidated (Hammond et al. 1989; Tabler et al. 1992). A key question that remains to be answered is whether single or mul- tiple sites are used for processing in vivo.
Weak self-cleavage of PSTVd RNAs has been reported by some researchers (Robertson et al. 1985) but not by others (Tabler and Sänger 1985; Tsagris et al. 1987a,b). It generally is thought that a cellular RNase which remains to be identified catalyzes the cleavage of concatemeric RNAs (Tsagris et al.
1987a,b). Reasoning that the general difficulty of demonstrat- ing self-cleavage of RNAs in Pospiviroidae could be attributed to the interference of nonribozyme RNA sequences in the sub- strates used during in vitro assays, Liu and Symons (1998)
Fig. 3. Asymmetric rolling circle replication of Potato spindle tuber viroid (PSTVd) and symmetric rolling circle replication of Avocado sunblotch viroid (ASBVd). The secondary structures of the genomic or circular RNAs are sketched to facilitate illustration of the approximate transcription initiation sites.