Analysis of 5’ and 3’ snoRNA termini maturation in - PowerPoint Presentation

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Analysis of 5’ and 3’ snoRNA termini maturation in Saccharomyces cerevisiae

Żaneta MatuszekInstitute of Genetics and Biotechnology University of Warsaw, PolandSupervisor: Prof. Joanna Kufel

7th Asia Pacific Biotech Congress 2015, Young Researchers Forum

Beijing, 13th of July 2015

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

snoRNA: structure and functions

7th Asia Pacific Biotech Congress 2015, Young Researchers ForumBox C/D snoRNA: 2’-

O-methylation (Me) of rRNA

box H/ACA snoRNA: pseudouridylation (

NΨ) of rRNA

CUGA

CUGA

UGAUGA

UGAUGA

Me

target RNA

target RNA

Me

Box C

Box C’

Box D’

Box D

5’

5’

5’

3’

3’

C/D

snoRNA

3’

ANANNA

ACA

N

Ψ

N

Ψ

Box H

H/ACA

snoRNA

Box ACA

NNN

5’

5’

3’

target RNA

Proteins

:

Fibrillarin/Nop1

N

op

56

N

op

58

15.5-kD

Proteins

:

Nhp2

N

op10

Cbf5 (dyskerin)

Gar1

Organization of snoRNA genes

monocistronic

yeast

plants

Metazoa

intronic

P

P

P

exon

exon

exon

yeast

animals

plants

policistronic

P

yeast

plants

independent genes

pre-mRNA introns

policistronic transcripts7th Asia Pacific Biotech Congress 2015, Young Researchers Forum

Model of snoRNA processing

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AAAAAAAAAAA

AAAAAAAAAAA

TRAMP:

nuclear surveillance

Tr

f4/5

+

A

ir1/2

+

M

tr4

poly(A)

polymerase

RNA binding

proteins

RNA DEVH helicaseSzczepaniak, not published

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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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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Materials (1): yeast strains used in the study

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 m

2,2,7G (TMG) cap of sn/snoRNA.

Cbp80 - 80 kDa nuclear cap-binding protein, both with Cbp20 are subunits of the cap-binding complex

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Strain

Genotype

References

BY 4741

MATa his3

Δ

1, leu2

Δ

0, met15

Δ

0, ura3

Δ

0

Euroscarf

BMA 64MATa, ura3-1, ade2-1, his3-11,15, trp1Δ, leu2-3,112, can1-100Baudin, 1993rnt1Δas BMA64 but RNT1::TRP1Chanfreau, 1998cbp80Δ MATa, ade2, ade3, his3, leu2-3, 112 rp1 ura3 CBP80::TRP1Fortes, 1999tgs1Δas BMA64 but TGS1:: HIS3Mouaikel, 2002rnt1Δ cbp80Δas rnt1Δ but CBP80::HIS3Szczepaniakrnt1Δ tgs1Δas rnt1Δ ale TGS1::HIS3Szczepaniakdcp2Δleu2-3112 his4-539 lys2-201 trp1 ura3-52 DCP2::TRP1Dunckley, 1999BMA64 + pRS415-snR68WTas BMA64 but pRS415-snR68WTSzczepaniakBMA64 + pRS415-snR68mutas BMA63 but pRS415-snR68mutSzczepaniakcbp80Δ + pRS415-snR68WTas cbp80Δ but pRS415-snR68WTSzczepaniakcbp80Δ + pRS415-snR68mutas cbp80Δ but pRS415-snR68mutSzczepaniaktgs1Δ + pRS415-snR68WTas tgs1Δ but pRS415-snR68WTSzczepaniaktgs1Δ + pRS415-snR68mutas tgs2Δ but pRS415-snR68mutSzczepaniakdcp2Δ + pRS415-snR68WTas dcp2Δ

but pRS415-snR68WTSzczepaniakdcp2Δ + pRS415-snR68mutas dcp2Δ but pRS415-snR68mutSzczepaniakDcp1/Dcp2 complex - responsible for rapid RNA decapping by removing the 5’ cap and leaving the 5’ end susceptible to exonucleolytic degradation

Materials (2):

snoRNA molecules under studysnR68snR64

snR65snR13 (control)

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Independently transcribed box C/D snoRNAs cleaved by Rnt1 (with a AGNN-capped stem-loop structure recognized by Rnt1)

Chanfreau et al. 2000

Independently transcribed

C/D box

snoRNA

with a TMG cap,

not processed at the 5’ end

Results

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5’ pre-snoRNA maturation defects lead to the accumulation 3’-extended precursors

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 (100-200 bp upstream 3’ end)

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2015, Young Researchers Forum

mature

snR68

rnt1Δ

wt

cbp80Δ

tgs1Δ

rnt1Δ cbp80Δ

rnt1Δ tgs1Δ

mature

snR64

3’-pre-snR68

3’-pre-snR64

oligo1

3’RNase HsnR135’5’3’

oligo2

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)

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Accumulation

of 3’-extended precursors

, not cleaved by Rnt1,

in strains with defects in 5’-end maturation. cRT-PCR analysisLigation after decapping by RNase H. Reverse trascription of circulated RNA (68RTLig or 68RTg)rnt1Δ tgs1Δ

wtcbp80Δ rnt1Δrnt1Δ cbp80Δ tgs1Δ200bp500bpsnR68 molecule; 68RTg and 68PCR 1FMature form: 136 bp

Dcp1/Dcp2 complex plays a role in pre-snoRNA processing – an alternative maturation pathway

Northern blot analysis for snR68, snR64 and snR65wt* – BY4741

25°C

transfered to 37°C for 1h

The effect is visibile only for snR68 – differences in the dependence on Rnt1 clevage?

Accumulation

of 3’-extended

precursors

in decapping mutants (

dcp1

or

dcp2

)

mature

snR68

3’-pre-snR68

(136

bp)

wt*

dcp1Δdcp2Δdcp1-2dcp1-2skiwt*dcp1Δdcp2Δdcp1-2dcp1-2skisnR13

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wt+snR68wt*

cbp80

Δ

+snR68wt

tgs1

Δ

+snR68wt

wt+snR68mut

cbp80

Δ

+snR68mut

tgs1

Δ

+snR68mut

wt*+snR68wt

dcp2

Δ

+snR68wt

wt*+snR68mutdcp2Δ+snR68mutsnR13Accumulation 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) rnt1Δ dcp1Δ and rnt1Δ dcp2Δ - lethal Accumulation of snR68 precursors in rnt1Δ and dcp2Δ GGAAA- UU- AG- CU- AU · GU · GG - CUACU · GG - C

C - GU - AG - CUA - UA - UC - GU - ACUG - CA - UC - GCGAU - AG - C5’ -- 87 nt - 3’AAA- UU- AG- CU- AU · GU · GG - CUACU · GG - CC - GU - AG - CUA - UA - UC - GU - ACUG - CA - UC - GCGAU - AG - C5’ -- 87 nt - 3’C

A

Not cleaved by Rnt1

mutation

pre-snR68

mature snR68

BMA6

GTGTTTCTGAAAGGGACCTTCAGGAGGTTACGATCAAGTATCTTGTGACATGCAAGAA 60 

rnt1

---TTTCTGAAAGGGACCTTCAGGAGGTTACGATCAAGTATCTTGTGACATGCAAGAA

55

cbp80

-----------------------------

ACGATCAAGTATCTTGTGACATGCAAGAA

29

(2

)

tgs1

------------------------------CGATCAAGTATCTTGTGACATGCAAGAA

28

 rnt1 GTGTTTCTGAAAGGGACCTTCAGGAGGTTACGATCAAGTATCTTGTGACATGCAAGAA 58 (2) -----------------CTTCAGGAGGTTACGATCAAGTATCTTGTGACATGCAAGAA 41 ----------------------GGAGGTTACGATCAAGTATCTTGTGACATGCAAGAA 36 -------------------------GGTTACGATCAAGTATCTCGTGACATGCAAGAA 33 -----------------------------ACGATCAAGTATCTTGTGACATGCAAGAA 29 ------------------------------CGATCAAGTATCTTGTGACATGCAAGAA 28 (2) WT -----------------CTTCAGGAGGTTACGATCAAGTATCTTGTGACATGCAAGAA 41 dcp1 ----------------------GGAGATTACGATTAAGTATCTTGTGACATGCAAGAA 36 (2) -------------------------GATTACGATTAAGTATCTTGTGACATGCAAGAA 33 -----------------------------ACGATTAAGTATCTTGTGACATGCAGGAA 29 (4) ------------------------------CGATTAAGTATCTTGTGACATGCAAGAA 287th Asia Pacific Biotech Congress 2015, Young Researchers ForumSequencing after cRT-PCR:~90% of precursors accumulated in dcp2Δ are not cleaved by Rnt1, but have shorter 5’ ends than observed in rnt1Δ strainsAlternative transtcription start site?Alternative decapping enzyme? 5’-end extensions in rnt1Δ and dcp2Δ strainlength (nt)number of clones

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 Rnt17th

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Acknowledgments

Joanna Kufel, Prof.Sylwia Szczepaniak, MScAnna Pastucha, PhDKarolina Stępniak, MSc And all other members of Kufel’s RNA lab

Thank you for your attention!

Literature

Chanfreau G, Legrain P, Jacquier A. (1998). Yeast RNase III as a key processing enzyme in small nucleolar RNAs metabolism. J. Mol. Biol. 284:975-988.

Chanfreau G, Buckle M, Jacquier A. (2000). Recognition of conserved class of RNA

tetraloops by Saccharomyces cerevisiae RNase III. Proc. Natl. Acad. Sci. U.S.A. 97(7):3142-7.

Grzechnik P, Kufel

J. (2008). Polyadenylation linked to transcription termination directs the processing of snoRNA precursors in yeast.

Mol. Cell

32:247-258.

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.

Kiss T. (2002). Small Nucleolar RNAs: An abundant group of noncoding RNAs with diverse cellular functions. Cell 109:145-148.Kuehner JN, Pearson EL, Moore C. (2011). Unravelling the means to an end: RNA polymerase II transcription termination. Nat. Rev. Mol. Cell. Biol.

12:283-294. LaCava J, Houseley J, Saveanu C, Petfalski E, Thompson E, Jacquier A, Tollervey D. (2005). RNA degradation by the exosome is promoted by a nuclear polyadenylation complex. Cell 121:713-24. Lee CY, Lee A. i Chanfreau G. (2003). The roles of endonucleolytic cleavage and exonucleolytic digestion in the 5’-end processing of S. cerevisiae box C/D snoRNAs. RNA 9:1362-1370.Matera AG, Terns RM, Terns MP. (2007). Non-coding RNAs: lessons from the small nuclear and small nucleolar RNAs. Nat. Rev. Mol. Cell. Biol. 8:209-220 Mouaikel J, Verheggen C, Bertrand E, Tazi J, Bordonne R. (2002). Hypermethylation of the cap structure of both yeast snRNAs and snoRNAs requires a conserved methyltransferase that is localized to the nucleolus. Mol. Cell 9:891-901.Vasiljeva L, Kim M, Mutschler H, Buratowski S, Menhart A. (2008). The Nrd1-Nab3-Sen1 termination complex interacts with the Ser5-phosphorylated RNA polymerase II C-terminal domain. Nat. Struct. Mol. Biol. 15(8):795-804.Samarsky, D.A. and Fournier, M.J. (1999). A comprehensive database for the small nucleolar RNAs from Saccharomyces cerevisiae. Nucleic Acids Res 27: 161–164.7th Asia Pacific Biotech Congress 2015, Young Researchers Forum