Isolation and Identification of the Cell-Stress Inducing snoRNA

Jarius Anderson-Baylor and Jean Schaffer

Center for Cardiovascular Research, Washington University School of Medicine

Mammalian adipose cells have the capacity to store large amounts of lipids that enter the body through consumption.  When non-adipose cells are exposed to high concentrations of lipids, they show signs of cellular stress, which commonly leads to lipotoxicity.  Chinese hamster ovary cells are one of the cells, which induce ER stress and oxidative stress when exposed to palmitate. In order to identify the cellular mediators of lipotoxicity within non-adipose cells, the Schaffer Lab conducted a genetic screen to isolate mutant Chinese hamster ovary (CHO) cells, which showed resistance to lipotoxic cell death. They then used promoter trap mutagenesis to generate the mutant CHO cells.  They found that one of the mutant palmitate resistant cells, named 6F2, has an insertion leading to one of two alleles, which results in decreased expression of the ribosomal protein L13a’s mRNA and protein synthesis. The L13a gene contains four different small nucleolar RNA (snoRNA) within the four introns of the pre-mRNA sequence.  When the genomic L13a sequence is expressed in 6F2 cells, this results in oxidative stress. However, expression of an L13a mRNA sequence does not result in oxidative stress due to palmitate sensitivity. We deduce, therefore, that the exons that make up the mRNA sequence are not responsible for the oxidative stress, and we hypothesize that one of the four introns containing snoRNA’s is responsible for the oxidative stress.  To test our hypothesis I set up an experiment in which I would isolate a single intron (intron 2) containing the desired snoRNA strand from the L13a genomic sequence.  I used a vector with which to contain and express the separated snoRNA: pcDNA 3.1 (+).  Since this vector does not have any restriction sites that match with those of the snoRNA, I chose to use a second vector, pSL301, to act as a connection between the two sequences.  I cloned ‘intron 2’ into the pSL301 vector with restriction enzymes Msc I and BsiW I; I then digested this new sequence, along with the pcDNA 3.1 (+) vector with two other restriction enzymes: Not I and Hind III.  Once ‘intron 2’ was isolated from the pSL301 vector, I cloned it into the desired vector, pcDNA 3.1 (+).  After the cloning, we transfect this DNA into the Chinese hamster ovary cells to test if they show any resistance to ER and oxidative stress.  Now that this new sequence has been transfect into a non-adipose cell, we may see if this specific snoRNA is responsible for the oxidative and ER stress found among CHO cells