Gene Deletion Strategy: Identify Function of Non-Coding RNA

Molecular Biology mainly Non coding RNA Project

Contents (Jump to)

Non coding RNA

Small Nuclear RNA genes

Materials and Methods

Media

YPD and YPD+G418 plates

Strain confirmation

References

A Gene Deletion Strategy to Identify the Function of Non-Coding RNA in the Eukaryotic Genome, Using the Model Organism Saccharomyces cerevisiae
Abstract

The yeast Saccharomyces cerevisiae is a key model organism for investigating cellular processes. While it is important to determine the role protein-coding genes play in biological, it is now clear that RNA also plays an important role in these cellular processes. Deletion cassettes were constructed successfully by a PCR based strategy for 12 ncRNAs, which are snR19, snR20, snR14, snR6, snR7, TLC1, SCR1, NME1, RPR1, RUF20, RUF21, and RUF22. Deletions cassettes were transformed into the diploid reference strain BY4743 and the ncRNA gene replaced with the KanMX marker byhomologous recombination. Heterozygous deletion strains were confirmed for each of the 12 genes by extensive PCR analysis and showed positive results for the insertion of the cassette. Following sporulation and tetrad dissection to segregate haploid spores and growth on different selective media, 10 of the 12 ncRNAs were shown to be essential for cell survival by producing 2 viable spores. These include snR19, snR20, snR14, snR6, snR7, TLC1, SCR1, NME1, RPR1 and RUF20.However, RUF21 and RUF 22 produced four viable haploid spores indicating that these are non-essential genes for the cell.This resource will now enable the role of ncRNAs in cellular processes to be determined, by growth of these deletion strains in a range of different conditions and in different genetic backgrounds.

Non coding RNA

The term non-coding RNA (ncrna) is usually utilized for RNA that does not encode a protein, however this does not imply that such Rnas don’t contain data nor have capacity. In spite of the fact that it has been by and large accepted that most hereditary data is transacted by proteins, late proof proposes that most of the genomes of vertebrates and other complex life forms is indeed translated into ncrnas, a considerable lot of which are alternatively joined and/or transformed into more diminutive items. These ncrnas incorporate micrornas and snornas (numerous if not a large portion of which stay to be distinguished), and likely different classes of yet-to-be-found little administrative Rnas, and a huge number of longer transcripts (counting complex examples of intertwining and covering sense and antisense transcripts), the vast majority of whose capacities are obscure. These Rnas (counting those inferred from introns) seem to embody a shrouded layer of internal signs that control different levels of quality articulation in physiology and advancement, including chromatin structural engineering/epigenetic memory, translation, RNA grafting, altering, interpretation and turnover. RNA administrative systems may focus a large portion of our complex attributes, assume a critical part in ailment and constitute an unexplored universe of hereditary variety both inside and between species. (Mattick JS, 2006)

In this project, the functionality of the following non-coding RNA genes in S.cerevisiae will be determined. The non-coding RNA genes mentioned in this study have significant importance in the yeast genome and the determination of functionality of these genes will unveil their exact contribution to eukaryotic cellular function. Non coding RNA genes are those that are transcribed but not translated. They code for either long (>200) or short (200<) non coding RNA. The main classes of non coding RNA are – Short: miRNA, snRNA, snoRNA, piRNA and tRNA, Long: antisense ncRNA, eRNA, meRNA, intergenicncRNA, psuedogenencRNA and 3’UTR ncRNA (Kowalczyket al., 2012). The highly abundant short non coding RNA genes have been well characterized and are known to play an important role in various cellular functions such as transcriptional regulation, translation regulation, RNA editing etc. Both long and short non coding RNAs are mostly transcribed by RNA polymerase II. However, some species of non coding RNAs are also transcribed by RNA pol III and I.

Small Nuclear RNA genes

The review article by Guthrie explains the steps that were undertaken to identify the snRNAs associated with the spliceosome (Guthrie and Patterson, 1988) and work carried out by the same group revealed that many of the ‘U-RNAs’ were encoded by single-copy genes, such as U5 which is encoded by snR7 (Riedel et al., 1986).

‘U’ in RNA stand for Uracil, each nucleotide in RNA encloses a ribose sugar, with carbons numbered 1? throughout 5?. A base is appended to the 1? position, all in all, cytosine (C), adenine (A), uracil (U), or guanine (G). Cytosine and uracil are pyrimidines, Adenine and guanine are purines.

Materials and Methods
Media
YPD and YPD+G418 plates

YPD media were made from 2% D-glucose, 1% Bacto yeast extract, 2% Bacto-peptone. YPD plates were prepared by adding 2% agar to the YPD media.

Agar (2%) and G418 Geneticin (200µg/ml) was supplement in the YPD media for the preparation YPD+G418 plates. The recepie for YPD+G418 plates is:

Bacto Peptone Difco 10g

Bacto Yeast Extract Difco 10g

Glucose 20g

Bacto Agar (if plates) 20g

Distilled water qsp 1L

Autoclave at 0,5bar

After autoclavage let cool down until 55°C

Add G418 to have a final concentration of 200mg/L

(1 mL of 200mg/mL G418 in 1L of YPD) (Genetics of Intra Specie Variations)

Identification of mating types and cross mating

Using 96 well plate, colonies of G418 resistance spores, which have been isolated from replica platting method, were inoculated in 100ul of YPD and 7ulof DMSO and incubated overnight at 30°C. This stock can be stored at -20°C.

The RT2 a strain and RT3 ? strain were allowed to grow in 5ml YPD culture overnight at 30°C. Following that two separated 96 well plates were used by filling each well with 10µl of the RT2 a strain or RT3 ? strain separately. Then 10µl of the tested samples were added each well of the known strains. These were then incubated overnight at 30oC.

Following incubation, metal frogger was used to replica plate the crosses onto deficiency plates without leucine and tryptophan. The incubation period was allowed until the colonies were able to be obtained on the deficient media.

For crossing, 2ul of the stock spores were pipetted on to deficiency plates without methionine and deficient plates without lysine. This was incubated at 30°C until the growth was determined.

When all data of the deficiency plates and mating types was known, cross spores of opposite mating type and one defficiency was done by adding 10ul of each mating stock culture into an eppendrof tube and incubated overnight at 30°C.

Strain confirmation

To confirm that the picked cells were haploid spores and that the cells were originally BY4743 yeast, all dissected spores were grown on different SD media.

RUF22

G418

-URA

-HIS

-LEU

–LYS

-MET

1A

+

–

–

–

+

+

1B

+

–

–

–

–

–

1C

–

–

–

–

–

–

1D

–

–

–

–

+

+

2A

+

–

–

–

+

+

2B

–

–

–

–

+

+

2C

–

–

–

–

–

–

2D

+

–

–

–

–

–

3A

–

–

–

–

+

+

3B

–

–

–

–

–

+

3C

+

–

–

–

–

–

3D

+

–

–

–

+

–

4A

–

–

–

–

+

+

4B

–

–

–

–

–

–

4C

+

–

–

–

–

+

4D

+

–

–

–

+

–

5A

–

–

–

–

+

–

5B

+

–

–

–

–

–

5C

–

–

–

–

–

+

5D

+

–

–

–

+

+

6A

–

–

–

–

+

+

6B

+

–

–

–

–

+

6C

+

–

–

–

–

–

6D

–

–

–

–

+

–

7A

–

–

–

–

–

+

7B

+

–

–

–

–

–

7C

–

–

–

–

+

+

7D

+

–

–

–

+

–

Table 2: Growth of the deletion strain tetrads on different SD media for RUF22 gene. Each colony was scored for significant growth (+) or no/poor growth (-) on each medium.

RUF21

G418

-URA

-HIS

-LEU

–LYS

-MET

1A

+

–

–

–

–

–

1B

–

–

–

–

–

+

1C

–

–

–

–

+

+

1D

+

–

–

–

+

–

2A

–

–

–

–

–

+

2B

+

–

–

–

+

–

2C

+

–

–

–

+

–

2D

–

–

–

–

–

+

3A

+

–

–

–

–

+

3B

–

–

–

–

–

+

3C

–

–

–

–

+

–

3D

+

–

–

–

+

–

4A

+

–

–

–

+

–

4B

+

–

–

–

–

+

4C

–

–

–

–

+

+

4D

–

–

–

–

–

–

5A

–

–

–

–

–

+

5B

+

–

–

–

+

–

5C

–

–

–

–

–

–

5D

+

–

–

–

+

+

6A

–

–

–

–

+

+

6B

+

–

–

–

+

–

6C

–

–

–

–

–

+

6D

+

–

–

–

–

–

Table 3: Growth of the deletion strain tetrads on different SD media for RUF21 gene. Each colony was scored for significant growth (+) or no/poor growth (-) on each medium.

TLC1

G418

-URA

-HIS

-LEU

–LYS

-MET

1A

+

–

–

–

–

+

1B

+

–

–

–

+

–

1C

–

–

–

–

–

–

1D

–

–

–

–

+

+

2A

–

–

–

–

+

+

2B

+

–

–

–

+

+

2C

–

–

–

–

–

–

2D

+

–

–

–

–

–

3A

+

–

–

–

+

+

3B

+

–

–

–

–

–

3C

–

–

–

–

–

+

3D

–

–

–

–

+

–

4A

–

–

–

–

+

+

4B

–

–

–

–

–

+

4C

+

–

–

–

–

–

4D

+

–

–

–

+

–

5A

–

–

–

–

–

–

5B

+

–

–

–

+

–

5C

–

–

–

–

–

+

5D

+

–

–

–

+

+

6A

+

–

–

–

+

–

6B

+

–

–

–

–

+

6C

–

–

–

–

–

–

6D

–

–

–

–

+

+

7A

–

–

–

–

–

–

7B

–

–

–

–

–

–

7C

+

–

–

–

+

+

7D

+

–

–

–

+

+

Table 4: Growth of the deletion strain tetrads on different SD media for TL gene. Each colony was scored for significant growth (+) or no/poor growth (-) on each medium.

The data in Tables 2 and 3 shows the results of the dissected RUF22 and RUF21 spores after being given the required time to grow. It was found that there was no growth in the SD(-URA), SD(-HIS) and SD(-LEU) plates indicating that they were BY4743-derived tetrads. Moreover, the growth of the spores observed in the YPD+G418, SD(-MET) and SD(-LYS) in the case of RUF22 and RUF21 deleted strains indicating that these cells derived from tetrads and were haploid knockouts.

References

Genetics of Intra Specie Variations. (n.d.). Retrieved September 19, 2014, from http://www.ens-lyon.fr/LBMC/gisv/index.php/en/protocols/yeast-methods/72-yeast-media-recipes

Mattick JS, M. I. (2006, April 15). Pub Med. Retrieved September 19, 2014, from http://www.ncbi.nlm.nih.gov/pubmed/16651366