Team:Fudan D/Ribozyme

In our project, we want to investigate the telomere’s shortening effect in yeast and try to take advantage of this phenomenon (also known as the aging effect since we are clear that the length of telomere will diminish after generations) to regulate the expression of the certain gene in aim of achieving some special changes in yeast cell. For example, yeast can “light up” itself automatically in several days if they can express GFP after twenty generations. Therefore, we hope that the telomere can function like a switch that turns up the light when it becomes shorter. However, it is known that telomere itself can’t work as a switch because it doesn’t turn on or off the expression of the flanking gene. As a result, it is necessary to combine telomere and another device that can directly control the gene expression to obtain the expected result. Under this circumstance, we incorporate the telomere and hammerhead ribozyme as a unit in attempt to construct a switch that can initiate the expression of upstream reporter gene.

In eukaryotes, several mechanisms have already been identified that can regulate the gene expression, including the strategies in DNA and DNA levels. In RNA level, some ribozymes, or RNA enzymes, are often employed to control the translation of mRNA in cytoplasm. Ribozymes have well-defined secondary and tertiary structures that enable them to perform certain chemical reactions. Among them, the hammerhead ribozyme is used in gene expression because of its cleaving property. Hammerhead ribozyme is a small, self-cleaving motif composed of a three-helical junction with a core of invariant nucleotides required for activity (M. Martick et al.) (Fig.1). After forming the specific secondary structure shown in Fig. 1, the enzyme region of hammerhead ribozyme will cleave the substrate region at the site with small hollow triangle.

There are two major characteristics in the sequence of the hammerhead ribozyme. Firstly, only about 15 nucleotides are immutable and essential to the activity of ribozyme, and the rest of the sequences of ribozyme can be altered as long as they satisfy the base-pairing principle. Secondly, it is possible to insert a sequence of hundreds of nucleotides between the substrate region and enzyme region of the ribozyme (Fig. 1). These characteristics are crucial to our design, and their importance will be explained in the following parts.

Many researches conducted in either mice or yeasts have indicated that hammerhead ribozymes have self-cleaving property. S. Meaux et al. reported that when a hammerhead ribozyme is located at the downstream of PtnA, it will cleave itself efficiently in vivo. The 5’ cleavage product of hammerhead ribozyme is very unstable because the poly(A) tail served as the protective cap at 3’ end is removed along with the 3’ cleavage product, thus the 5’ cleavage products will disappear with a half life of approximately 1 minute after transcription is shut off. Therefore, this mechanism can reduce the translation of the gene at the upstream of the ribozyme to a great extent.

We designed a model in order to fulfill the goal of turning on a gene of the yeast after generations. The model consists of several parts, including ADH4 (used in recombination between the plasmid and chromosome VII of yeast), a marker (used to screen the yeasts containing the given plasmid), a promoter, a reporter gene (used to indicate the change in phenotype of the engineered yeast), and a unit of ribozyme and telomeres as a switch of the upstream reporter gene (Fig.2).

At the first few generations, the enzyme region of the ribozyme remains complete in yeast. After the transcription and the export of mRNA, the enzyme region of ribozyme will bind to the substrate region, form a secondary structure, and subsequently cleave the substrate region at the specific site. Since the 5’ cleavage product doesn’t have poly(A) tail, it will soon be degraded in cytoplasm. Accordingly, the translation of the reporter gene will be blocked.

After several generations, the length of telomere will gradually decrease, until the terminal telomere is totally cut off. However, due to the fact that the telomere between the two regions of ribozyme is still too long, the cleaving process continues and part of the enzyme region starts to be cut off because it is at the terminal of the chromosome. If the enzyme region is damaged, it can no longer work as an effective ribozyme to cleave the substrate region. Consequently, the translation of reporter gene will not be blocked by the degradation of mRNA. Therefore, if we measure the level of the protein and find that there is significant difference between the amount of protein in first few generations and later generations, we can guarantee that telomere’s shortening effect does act as a switch to initiate the expression of reporter gene.

When we were designing the model, a plenty of factors should be taken into consideration. First, since the overelongated telomere leads to the DNA cleavage from the 3’ end, we need to find a device in form of nucleotides that located in DNA. This device can help to regulate the expression of flanking gene, no matter in the level of DNA or RNA, and the removal of part of the device will lead to the total loss of its function as a regulator. Second, if the device is the binding site of a specific protein that regulates the gene expression, we have to ensure that the ligand (the specific protein) exists in the yeast cell, or the device will be futile. Third, the length of the device must be less than 50 bp. The telomere of yeast chromosome usually has the length of 325±75 bp (A. Ray and K. W. Runge). The reason why the telomere shortens from the first generation is that the yeast cell counts the length of the whole telomere simply by adding up the lengths of two separated telomeres, and the total length is larger than 400 bp (600+50>400). However, if the spacer (non-telomeric sequence) between two telomeres is too long (larger than 50 bp), the yeast cell may not count in the telomere far away from the terminal of chromosome, and telomerase will start to extend the telomere because it is too short “in yeast’s mind” (A. Ray and K. W. Runge).

Based on the criteria mentioned above, hammerhead ribozyme is one of the best devices we can find. The activity of hammerhead ribozyme depends on several essential nucleotides. As long as the nucleotides are cleaved, ribozyme loses most of the activity. Moreover, ribozyme itself acts as an enzyme, which means that it doesn’t rely on other proteins to regulate gene expression. Last but not least, the enzyme region of hammerhead ribozyme is about 40 nucleotides, which will not influence the counting of telomere.

[1] M. Martick et al., A discontinuous hammerhead ribozyme embedded in a mammalian messenger RNA. Nature (2008); 454(7206): 899-902.

[2] S. Meaux et al., Yeast transcripts cleaved by an internal ribozyme provide new insight into the role of the cap and poly(A) tail in translation and mRNA decay. RNA (2006); 12: 1323-1337.

[3] A. Ray and K. W. Runge. The yeast telomere length counting machinery is sensitive to sequences at the telomere-nontelomere junction. Molecular and Cellular Biology (1999); 19(1): 31-45.