1. Bio Asian Pacific 2015, Young Researchers Forum
7th Asia Pacific Biotech Congress 2015, Young Researchers Forum
Beijing, 13th of July 2015
Analysis of 5’ and 3’ snoRNA termini maturation in Saccharomyces cerevisiae
Żaneta Matuszek
Institute of Genetics and Biotechnology
University of Warsaw, Poland
Supervisor: Prof. Joanna Kufel

2. 7th Asia Pacific Biotech Congress 2015, Young Researchers Forum
Presentation agenda
Introduction to snoRNA biology
structure and function of snoRNA
snoRNA genes organization
snoRNA processing model
Aims of the study
Materials
strains characterization
snoRNA molecules
Results
Conclusions
7th Asia Pacific Biotech Congress 2015, Young Researchers Forum

3. snoRNA: structure and functions
Bio Asian Pacific 2015, Young Researchers Forum
snoRNA: structure and functions
Box C/D snoRNA: 2’-O-methylation (Me) of rRNA
box H/ACA snoRNA: pseudouridylation (NΨ) of rRNA
CUGA
UGAUGA Me
target RNA
Box C
Box C’
Box D’
Box D 5’ 3’
C/D
snoRNA
Proteins:
Fibrillarin/Nop1
Nop56
Nop58
15.5-kD 3’
ANANNA
ACA NΨ
Box H
H/ACA
snoRNA
Box ACA
NNN 5’
target RNA
Proteins:
Nhp2 
Nop10
Cbf5 (dyskerin)
Gar1
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4. Organization of snoRNA genes
monocistronic
yeast
plants
Metazoa
intronic P
exon
animals
policistronic
independent genes
pre-mRNA introns
policistronic transcripts
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5. Model of snoRNA processing
Bio Asian Pacific 2015, Young Researchers Forum
Model of snoRNA processing
AAAAAAAAAAA
Assessment of the transcript kinetics allows us to conclude that snoRNAs are processed from polyadenylated precursors by independent termination at site I or II, immediately followed, either co- or posttranscriptionally, by the addition of poly(A) tails at both terminators. In wild-type cells, snoRNAs are generated from either precursor but preferably from polyadenylated site I-associated transcripts processed by Rrp6 to M∗ intermediates, which in turn are trimmed to mature species, also by Rrp6. oligoadenylation, in the poly(A) tail synthesis of site I transcripts and, when Trf4 is missing, by shifting termination to site II that is polyadenylated by another polymerase.
Trf4/5 polymerases are not involved in the addition of poly(A) tails at terminator II but may well act at site I. Trf4 may also contribute to the synthesis of major precursors independently of its polymerase activity
Pap1 participates in polyadenylation of pre-snoRNAs, particularly following termination at site II.
The length of the poly(A) tail at terminator I (∼80 nt) and the analysis of pap1 mutants point to the activity of Pap1 also at this site. We envisage that Trf4/5 may initiate the process by addition of short oligo(A) tails that are further extended by Pap1 to ensure effective recruitment of exonucleases. This hypothesis would explain how Pap1 recognizes substrates terminated by the Nrd1/Nab3 complex that has no apparent connection with Pap1 but interacts with Trf
Szczepaniak, not published
TRAMP: nuclear surveillance
Trf4/ Air1/ Mtr4
poly(A)
polymerase
RNA binding
proteins
RNA DEVH
helicase
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6. Coupling of 5’ and 3’ snoRNA end formation in yeast
Factors involved in transcription termination and formation of mRNA 3’ end associate with the promoter region – coupling of transcription and mRNA ends processing [Topisirovic, 2011]
Cap-binding and Nrd1/Nab3/Sen1 complexes copurify, suggesting interaction of machineries acting on both snoRNA ends [Vasiljeva, 2006]
Interaction between CBC and Nrd1-Nab3 is direct [Szczepaniak, not published]
CBC remains associated at snoRNA genes until transcription termination [Szczepaniak, not published]
Rnt1 is recruited to maturing snoRNAs at late stages of transcription [Szczepaniak, not published]
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7. 7th Asia Pacific Biotech Congress 2015, Young Researchers Forum
Aim of the study
Hypothesis:
Processing of 5’ and 3’ snoRNA ends in Saccharomyces cerevisiae is coupled.
Analysis of snoRNA 3’ and 5’ end status in mutants with defective end formation: cRT-PCR analysis
Description of snoRNA synthesis defects in mutant strains: northern blot analysis.
Characterization of an alternative 5’ pre-snoRNA formation mechanism by Dcp1/Dcp2-dependent cap hydrolysis in the absence of Rnt1 cleavage: nothern blot analysis.
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8. Materials (1): yeast strains used in the study
Bio Asian Pacific 2015, Young Researchers Forum
Materials (1): yeast strains used in the study
Strain
Genotype
References
BY 4741
MATa his3Δ1, leu2Δ0, met15Δ0, ura3Δ0
Euroscarf
BMA 64
MATa, ura3-1, ade2-1, his3-11,15, trp1Δ, leu2-3,112, can1-100
Baudin, 1993
rnt1Δ
as BMA64 but RNT1::TRP1
Chanfreau, 1998
cbp80Δ
MATa, ade2, ade3, his3, leu2-3, 112 rp1 ura3 CBP80::TRP1
Fortes, 1999
tgs1Δ
as BMA64 but TGS1:: HIS3
Mouaikel, 2002
rnt1Δ cbp80Δ
as rnt1Δ but CBP80::HIS3
Szczepaniak
rnt1Δ tgs1Δ
as rnt1Δ ale TGS1::HIS3
dcp2Δ
leu his4-539 lys2-201 trp1 ura3-52 DCP2::TRP1
Dunckley, 1999
BMA64 + pRS415-snR68WT
as BMA64 but pRS415-snR68WT
BMA64 + pRS415-snR68mut
as BMA63 but pRS415-snR68mut
cbp80Δ + pRS415-snR68WT
as cbp80Δ but pRS415-snR68WT
cbp80Δ + pRS415-snR68mut
as cbp80Δ but pRS415-snR68mut
tgs1Δ + pRS415-snR68WT
as tgs1Δ but pRS415-snR68WT
tgs1Δ + pRS415-snR68mut
as tgs2Δ but pRS415-snR68mut
dcp2Δ + pRS415-snR68WT
as dcp2Δ but pRS415-snR68WT
dcp2Δ + pRS415-snR68mut
as dcp2Δ but pRS415-snR68mut
Rnt1 – homologous to bacterial Rnase III, double-strand-specific endoribonuclease, functions in the 5’-end processing of some C/D box snoRNA, substrates are capped by tetraloops with the consensus AGNN sequence.
Tgs1 – nuclear trimethyl guanosine synthase I, responsible for m7G RNA cap hypermethylation to m2,2,7G (TMG) cap
of sn/snoRNA.
cleaves double-stranded structures capped by tetraloop with the sequence AGNN, and the enzyme selects the phosphodiester bond to be cleaved between 13 and 16 bp from the tetraloop (Chanfreau et al. 2000). Independently transcribed c/d box snoRNA, also two located in the introns
Cbp kDa nuclear cap-binding protein, both with Cbp20 are subunits of the cap-binding complex
Dcp1/Dcp2 complex - responsible for rapid RNA decapping by removing the 5’ cap and leaving the 5’ end susceptible to exonucleolytic degradation
7th Asia Pacific Biotech Congress 2015, Young Researchers Forum

9. Bio Asian Pacific 2015, Young Researchers Forum
Materials (2): snoRNA molecules under study
Independently transcribed box C/D snoRNAs cleaved by Rnt1 (with a AGNN-capped stem-loop structure recognized by Rnt1)
snR68
snR64
snR65
Mapy snR64, 68, 65
Independently transcribed C/D box snoRNA with a TMG cap, not processed at the 5’ end
snR13 (control)
Chanfreau et al. 2000
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10. 7th Asia Pacific Biotech Congress 2015, Young Researchers Forum
Results
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11. Bio Asian Pacific 2015, Young Researchers Forum
5’ pre-snoRNA maturation defects lead to the accumulation 3’-extended precursors
mature snR68
rnt1Δ wt
cbp80Δ
tgs1Δ
rnt1Δ cbp80Δ
rnt1Δ tgs1Δ
mature snR64
3’-pre-snR68
3’-pre-snR64
RNase H
oligo1 3’ 5’ 3’
oligo2 5’
Northern blot analysis for snR68 and snR64 molecules in wt and mutant strains.
Hybridization after RNase H treatment in the presence of oligo complementary to the mature snoRNA ( bp upstream 3’ end)
– immature sequences only at 3’ end. Loading znaleźć
snR13
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12. Deficiency of 5’ end maturation affects the 3’ pre-snoRNA end status
snR68
136 bp
Rnt1
68HLig
68PCR 2R
68RTg (1R)
Sn68pre5
68RTLig
68PCR 1F
68PCRlig (2F)
cRT-PCR analysis
Ligation after decapping by RNase H. Reverse trascription of circulated RNA (68RTLig or 68RTg)
rnt1Δ tgs1Δ wt
cbp80Δ
rnt1Δ
rnt1Δ cbp80Δ
tgs1Δ
200bp
500bp
snR68 molecule; 68RTg and 68PCR 1F
Mature form: 136 bp
Accumulation of 3’-extended precursors, not cleaved by Rnt1, in strains with defects in 5’-end maturation.
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13. Bio Asian Pacific 2015, Young Researchers Forum
Dcp1/Dcp2 complex plays a role in pre-snoRNA processing – an alternative maturation pathway
Accumulation of 3’-extended precursors in decapping mutants (dcp1 or dcp2)
mature snR68
3’-pre-snR68
(136 bp)
wt*
dcp1Δ
dcp2Δ
dcp1-2
dcp1-2ski
snR13
Northern blot analysis for snR68, snR64 and snR65
wt* – BY4741
25°C
transfered to 37°C for 1h
dcp1-2 – termosensitive strain
Ski complex - in yeast guides RNA to the exosome complex for degradation, consists of three main proteins Ski 2,3 and 8.
The effect is visibile only for snR68 – differences in the dependence on Rnt1 clevage?

14. Bio Asian Pacific 2015, Young Researchers Forum
Accumulation of snR68 precursors in rnt1Δ and dcp2Δ
rnt1Δ dcp1Δ and rnt1Δ dcp2Δ - lethal
wt+snR68wt*
cbp80Δ+snR68wt
tgs1Δ+snR68wt
wt+snR68mut
cbp80Δ+snR68mut
tgs1Δ+snR68mut
wt*+snR68wt
dcp2Δ+snR68wt
wt*+snR68mut
dcp2Δ+snR68mut
snR13 G A
- U U
- A
- C
U · G
G - C C
C - G
U - A
A - U
5’ -
- 87 nt - 3’
Not cleaved by Rnt1
mutation
pre-snR68
Schemat mutacji; All substrates of yeast RNase III (Rnt1p) are capped by terminal tetraloops showing the consensus AGNN and located within 13–16 bp to Rnt1p cleavage sites. We show that these tetraloops are essential for Rnt1p cleavage and that the distance to the tetraloop is the primary determinant of cleavage site selection. 
mature snR68
Accumulation of precursors in strains with pRS415-snR68wt/mut (mutation in the Rnt1 recognition motif: AGGAACAA).
BMA64 (wt for Rnt1 mutants) and BY4741 (wt* for Dcp1/Dcp2 mutants)
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15. 5’-end extensions in rnt1Δ and dcp2Δ
length
(nt)
number of clones
strain
Sequencing after cRT-PCR:
~90% of precursors accumulated in dcp2Δ are not cleaved by Rnt1, but have shorter 5’ ends than observed in rnt1Δ strains
Alternative transtcription start site?
Alternative decapping enzyme?
BMA GTGTTTCTGAAAGGGACCTTCAGGAGGTTACGATCAAGTATCTTGTGACATGCAAGAA
rnt TTTCTGAAAGGGACCTTCAGGAGGTTACGATCAAGTATCTTGTGACATGCAAGAA
cbp ACGATCAAGTATCTTGTGACATGCAAGAA (2)
tgs CGATCAAGTATCTTGTGACATGCAAGAA
rnt1
GTGTTTCTGAAAGGGACCTTCAGGAGGTTACGATCAAGTATCTTGTGACATGCAAGAA (2)
CTTCAGGAGGTTACGATCAAGTATCTTGTGACATGCAAGAA
GGAGGTTACGATCAAGTATCTTGTGACATGCAAGAA
GGTTACGATCAAGTATCTCGTGACATGCAAGAA
ACGATCAAGTATCTTGTGACATGCAAGAA
CGATCAAGTATCTTGTGACATGCAAGAA (2)
WT CTTCAGGAGGTTACGATCAAGTATCTTGTGACATGCAAGAA
dcp1
GGAGATTACGATTAAGTATCTTGTGACATGCAAGAA (2)
GATTACGATTAAGTATCTTGTGACATGCAAGAA
ACGATTAAGTATCTTGTGACATGCAGGAA (4) CGATTAAGTATCTTGTGACATGCAAGAA
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16. 7th Asia Pacific Biotech Congress 2015, Young Researchers Forum
Conclusions
Defects of 5’ snoRNA end processing, especially lack of Rnt1 cleavage, lead to inefficient 3’ end formation and accumulation of extended precursors.
This phenotype is additionally modulated by mutations of other proteins acting at snoRNA 5’ ends: it is partly rescued by the absence of CBC and additionally strenghtened by deletion of Dcp1/Dcp2 or Tgs1
Synthesis of mature snoRNAs in the absence of Rnt1 cleavage suggests the existance of an alternative maturation pathway mediated by the Dcp1/Dcp2 complex and independent of Rnt1
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17. Acknowledgments Joanna Kufel, Prof. Sylwia Szczepaniak, MSc
Anna Pastucha, PhD
Karolina Stępniak, MSc
And all other members of Kufel’s RNA lab

18. Thank you for your attention!

19. 7th Asia Pacific Biotech Congress 2015, Young Researchers Forum
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Grzechnik P, Kufel J. (2008). Polyadenylation linked to transcription termination directs the processing of snoRNA precursors in yeast. Mol. Cell 32:
Kim M, Krogan NJ, Vasiljeva L, Rando OJ, Nedea E, Greenblatt JF, Buratowski S. (2004). The yeast Rat1 exonuclease promotes transcription termination by RNA polymerase II. Nature 432:517– 522.
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