RNA SPLICING AND RNA A TO I EDITING

Bachelor Thesisprepared by Mariya Licheva

17. 12. 2014

THE RNA SPLICING PROCESS

Important post-transcriptional process Process of modification of the nascent pre-

mRNA transcripts in which the introns are removed and exons are joined together

The Spliceosome is a large ribonucleoprotein complex that mediates the splicing process

Accuracy of the splicing process assures proper gene expression

THE SPLICEOSOME

highly dynamic RNP-Machine mediate two SN2 - transesterification reactions:

1) the 2' - OH ribose group of the branchpoint A attacks the 5' phosphate of the intron

2) the 3'-OH group of 5' exon ribose attacks the 5' phosphate of the 3'exon the Spliceosome is responsible for the removal of the majority

of precursor mRNA intron sequences the spliceosome consists of 5 uridine-rich RNAs, the so-called

small nuclear RNAs (snRNAs) - U1, U2, U4/U6, and U5 the snRNAs form complexes with a set of proteins. These RNA-

protein complexes are called snRNPs.

Coupling mRNA processing with transcription in time, and space , Reviews , David L.Bentley

THE FORMATION OF THE SPLICEOSOMAL COMPLEX IS A STEPWISE PROCESS

Active Spliceosome allows the first transesterification reaction ,

generating the C Complex

After the second catalytic reaction the mature RNA is released together with the

snRNPs, proteins and Lariat structure

The Spliceosome: Design Principles of a Dynamic RNP Machine : Markus C. Wahl,Cindy L. Will ,

Reinhard Lührmann

RNA EDITING

is a post-transcriptional process in which a genomically encoded nucleoside is changed to another nucleoside in RNA

The majority of RNA editing in mammals is either deamination of Cytosine (C) to Uridine (U) or Adenosine (A) to Inosine (I)

www.nobelprize.org

ADAR (ADENOSINE DEAMINASES ACTING ON RNA) ENZYMES

catalyze the RNA A to I editing of double stranded RNA molecules

Deamination of Adenosine to Inosine leads to nucleotide substitution, because the Inosine is recognized as Guanosine during cellular processes

ADAR mediated RNA editing is proven to have a key role in the brain development and its normal function

Improper regulation of the RNA A to I editing results in

- diseases of the central nervous system such as schizophrenia, ALS , epilepsy ...

- cancer

- wrong splicing products

Coupling mRNA processing with transcription in time, and space , Reviews , David L.Bentley

Exon Intron

HYPOTHESIS

The duration of the splicing process is positively correlated with the frequency of the A to I Editing in a single pre-mRNA molecule

The longer the splicing process takes, the more A will be edited to I

EXPERIMENTS :1)Testing how JetPEI and PEI influence the transfection process in HeLa and Hek cells and comparison of the toxicity of both transfection reagents.

2) Testing the splicing efficiency and A to I editing levels in three different Azin1 Plasmids : Azin1 -AdML, Azin1-ß-Globin, Azin - TJP1. 3) Determination of the A to I editing levels in HeK cells with Gria 2 Plasmids (AdML ; Ad3.1; Ad1.5) and additional extra cellular ADAR Enzyme (Co-Transfection).

COMPARISON BETWEEN THE INFLUENCE OF JETPEI AND PEI OF THE TRANSFECTION PROCESS IN HELA AND HEK CELLS

Transfection with JetPEI /PEI as a transfection reagent and a Plasmid containing a GFP - Construct

Two different kinds of cells (Hek cells and HeLa cells) were used Four different concentrations of JeTPEI and PEI were used in the transfection experiment :

- 1 microliter

- 1,5 microliter

- 3 microliter

- 6 microliter Treated cells were incubated at 37°C over night

COMPARISON BETWEEN THE INFLUENCE OF PEI AND JETPEI AS TRANSFECTION REAGENTS IN HEK AND HELA CELLS

Conclusion : JetPEI acts as a better transfection reagent in comparison to

PEI

Comparison in HeLa cells

Comparison in Hek cells

Trea

ted

with

JetP

EITre

ated

with

PEI

1µl 1,5µl 3µl 6µl

TOXICITY LEVELS OF JETPEI AND PEI

Question : How do different concentrations of JeTPEI and PEI influence the cell number during the transfection ?

Results:1) With increasing concentration of JetPEI and PEI as a transfection reagent the cell number decreases.

2) At lower concentration of PEI and JeTPEI , PEI is more toxic then JeTPEI

3) At high concentration both reagents show high level of toxicity

JETPEI AND PEI AS TRANSFECTION REAGENTS

U2OS cells treated with JetPEI after three days

U2OS cells treated with PEI after three days

U2OS cells - Ostreosarcoma cells

DETERMINING THE A TO I EDITING LEVELS AND THE SPLICING EFFICIENCY IN THREE AZIN1 CONSTRUCTS

Question : 1) How does the splicing efficiency change in three different constructs : Azin1 ß-Globin ,Azin1 AdML ,Azin1 TJP1 ?

2) How do the A to I editing levels change in these three constructs? Experimental procedure :1) Co-transfection with ADAR1 enzyme

2) Isolation of the RNA molecules from cells

3) Transformation of the RNA molecules into cDNAs with Reverse Transcription Method

4) PCR with the cDNA molecules

5) Gel-Extraction of the following fragments

6) Sequencing

ADAR1 AND ADAR 2 ENZYMES

there are three ADAR genes in mammals and two of them encode active deaminases - ADAR1 and ADAR2

due to alternative splicing process and alternative promotor regions there are two different protein forms of ADAR1 : ADAR1-p150 and ADAR1-p110

2011. George

Z-DNA binding motif

ds RNA binding motif

THE STRUCTURE OF AZIN1 CONSTRUCTS

Azin 1-AdML - shows excellent splicing efficiency Azin 1-ß-Globin - shows good splicing efficiency in comparison to Azin1-TJP1 and worst splicing

efficiency in comparison to Azin1-AdML Azin1-TJP1 has the worst splicing efficiency

The Azin constructs are linked to three different intron-exon sequences, which show different splicing efficiency

RESULTS : HOW DOES THE SPLICING EFFICIENCY CHANGE IN THE THREE MUTATED CONSTRUCTS ?

The splicing efficiency of the Azin-AdML construct is the greatest and for the Azin-TJP1 construct there is almost not splicing product detectable.

The Azin-ß-Globin construct exhibits some splicing efficiency, which is still significantly lower in comparison to the Azin-AdML construct.

AdM

L

ß-Gl

b

TJP1

pre-mRNAalternative splicing

productsmRNA

500 bp400bp300bp

200bp

100bp

RESULTS : HOW THE A TO I EDITING LEVELS CHANGE IN THE THREE CONSTRUCTS?

Azin1- AdML Azin1 -ß-Globin Azin1 -TJP1

There are no changes in the editing levels in the three constructs/ Editing is not detectable

DETERMINATION OF THE A TO I EDITING LEVELS IN THREE DIFFERENT GRIA2 CONSTRUCTS USING HEK CELLS AND EXTRA CELLULAR ADAR2 - ENZYME

Question : 1) How the mutations in the three Gria2 constructs influence the splicing process ?

2) Is there any correlation between the Splicing Duration and the amount of Editing in those constructs ? Hypothesis : The duration of the splicing process is positively correlated with the frequency of the

A to I Editing in the Gria2-constructs. Experimental procedure: 1) Transfection into Hek cells using three different Gria2 constructs : AdML , Ad3.1 and Ad 1.5

2) RNA- Isolation

3) Performing of Reverse Transcription and transforming the RNA molecules into cDNA molecules

4) PCR

5) Sequencing

GRIA2 CONSTRUCTS

Gria2-AdML - Wild type pre-mRNA Gria2-3.1 -pre-mRNA has four

mutations in the pyrimidine rich tract Gria2-1.5 - pre-mRNA has two

additional mutations in the pyrimidine rich tract

These mutations weaken the 3‘ splice site and should lead to reduced splicing efficiency

http://mol-biol4masters.masters.grkraj.org/html/RNA_Processing3A-Cis_Splicing_of_Pre_mRNAs.htm

U

Exon/Intron boundary

AdML

Ad3.1

Ad1.5

Gria2-Exon13 AdML-Exon2

RESULTS :

AdM

L

Ad3.

1

Ad1.

5

pre-mRNAAlternative splicing products

Alternative Splicing productsmRNA

500 bp 400 bp300 bp

200 bp

100 bp

1) The Splicing efficiency decrease from AdML to

Ad3.1

2) By Ad1.5 the mRNA is absent and two alternative

splicing products are present

RESULTS:

AdMl Ad3.1 Ad1.5

45,47%77,24%19,6%

1) There is an increase in the editing levels from AdML to Ad3.1 2) The Ad1.5 construct shows lower editing levels

CONCLUSION : THE DURATION OF THE SPLICING PROCESS IS POSITIVELY CORRELATED WITH THE FREQUENCY OF THE EDITING PROCESS IN THE GRIA2-CONSTRUCTS

OUTLOOK :

OUTLOOK :

Experimental procedure :

1) Standard - PCR Program2) Overlap PCR Program

• The production of three new Gria2 constructs with three different mutations in the pyrimidine rich tract will give us better overview on the coupling between splicing and editing.

• Ideally, we want to generate constructs that splice less efficiently than Gria2-AdML and more efficiently than Gria2-Ad3.1.

• In addition we want to avoid that alternatively spliced products occur.

Standard PCR

A1

A2

B1

B2 C2

C1 CBA

Overlap PCR

Fragments A1,B1 and C1 were made with a

normal Forward Primer and a mutated

Reverse Primer

Fragments A2 , B2 and C2 were made

with normal Reverse Primer and a mutated

Forward Primer

The mutation in all fragments occurs in the pyrimidine rich

tract

SUMMARY :

JetPEI acts as a better transfection reagent in comparison to PEI With increasing concentrations of JetPEI and PEI as a transfection reagent the cell number decreases. At lower concentration of PEI and JeTPEI , PEI is more toxic then JeTPEI At high concentration both reagents show high level of toxicity

Co-transfection experiment with ADAR1 enzyme shows no changes in the editing levels in the three Azin1 constructs

The duration of the splicing process in the three Gria2 constructs is positively correlated with the frequency of the editing process

The production of three new Gria2 constructs with three different single mutations in the pyrimidine rich tract will give us better overview on the coupling between splicing and editing.