Difference between revisions of "Part:BBa K1051900"

Revision as of 11:47, 28 September 2013

G1 phase magic with cell synchronization device and CRISPRi induced alternative splicing device.

clb5 +d-box +GFP + src1 intron + tom40 + cca + gal + dCas9 + cca + snr52 + sgRNA(Hub1) + sup4

Alternative Splicing Device

In yeast cells, alternative splicing is a common process in before gene transcription. The splicing sites are located in 5'UTR of introns and can by recognized and spliced by some splicesome. Alternative splicing can produce two isoforms from one gene, thus can be used in our project to monitor whether the Sic1 system work efficiently. Here we find a intron with alternative splicing sites in yeast cells named SRCI intron.

Src1 Intron and Hub1

SRC1 intron has two 5' splicing site whose efficiency is regulated by protein hub1. Originally, the existence of the intron would produce two mature mRNA in proportion. After engineering, the existence of protein hub1 regulates to the preservation of intron precisely, which would make the following mRNA be expressed normally or not. As for the silencing of wild type gene HUB1, we choose CRISPRi that is comparably easy to use reversibly.

Alternative splicing substantially increases the gene product diversity and is a major source of cell type differentiation. A good example is the alternative splicing of Saccharomyces cerevisiae SRC1 pre-mRNA, which is promoted by the conserved ubiquitin-like protein Hub1. It can function through binding non-covalently to a conserved element termed HIND in the spliceosomal protein Snu66. Such binding makes the splicesome target sites change and moderately alters spliceosomal interactions.

Hub1 is a ubiquitin-like modifier (UBL) that covalent modify the proteins. Interest enough, it harbors several different to other UBLs in which it possesses a C-terminal double tyrosine motif while others having a GG motif. The Snu66, a tri-snRNP in yeast spliceosome, possesses with two N-terminal HINDs (Hub1-INteraction Domain). The Hub1–HIND interaction comprises a strong salt bridge accompanied by several hydrophobic contacts and high affinity. Such binding modifies the spliceosome rather than modulating the properties of an individual binding partner. Hub1-controlled splicing occurs universally in eukaryotes. SR proteins and hnRNPs involved in spliceosome targeting do not seem to exist in S. Cerevisiae , and thus the Hub1-dependent mechanism may be evolutionarily older.

Scr1 is a protein in yeast having alternative splicing sites in its intron. The characteristic differential Hub1 dependence of SRC1 alternative splicing requires the tandem arrangement of overlapping 5’ splice sites. The Hub1 binding spliceosome can splice the intron from both downstream 5’ sites as well as the upstream 5’ sites with preference to the former one.

Figure. Principle for alternative splicing of Src1.

Showed in Figure.above When cutting in the upstream splice sites, the exons flanking around it would be translated as a fusion protein Scr-S. However, when cutting at the down stream one, the left 4bp in intron would result in a frame shift, thus only the forward exon can express named Scr-L.

dCas9 CRISPR interface system

CRISPR shorts for Clustered Regularly Interspaced Palindromaic Repeats system, which can be targeted to DNA using RNA, enabling genetic editing of any region of the genome in many organisms.(Cho, Kim, Kim, & Kim, 2013). In the type II CRISPR/Cas system, a ribonucleoprotein complex formed from a single protein (Cas9), a crRNA, and a trans-acting CRISPR RNA (tracrRNA) can carry out efficient crRNA-directed recognition and site-specific cleavage of foreign DNA(Deltcheva et al., 2011). After mutated the endonuclease domains of the Cas9 protein, it creates a programmable RNA-dependent DNA-binding protein. The sgRNA consists of three domains: a 20 nt complementary region for specific DNA binding, a 42 nt hairpin for Cas9 binding (Cas9 handle), and a 40 nt transcription terminator derived from S. Pyogenes. After translation, the Cas9 binds to sgRNA to form a protein-RNA complex, which can recognize target sites in the genome sequence and bind to it. Then, it could block RNA polymerase and transciption elongation.

Figure. sgRNA structure.

Figure. Principle of the function of dCAS9 and guide RNA.

Alternative Splicing by CRISPRi

To predict the alternative outcome, we also made an intron model to show different results due to incubating in different media. In our project, intron can be spliced in two different ways, providing a completely different outcome because of frame-shift, and this result is not a change like 1-0 to 0-1, but somehow more like a change between 0.4-0.6 and 0.8-0.2.

Reactions:

Parameter Table:

There are 4 parameters that we cannot find during our research, including kass, kdis representing the association and dissociation rate of CRISPRi system, and kass1 and kdis1 representing the association and dissociation rate of Hub1p and spliceosome.

We run parameter scan for each system individually, and found out that with CRISPRi system is more efficient with higher kass and lower kdis, as expected.

And about the alternative splicing model, we attempted to fit simulation result to experimental one. In Hub1 expressed system, L-mRNA will rise at first but descend to an equilibrium stage while S-mRNA will directly rise to its own equilibrium stage.

kass1 should be larger than kdis1, or L-mRNA will be produced more than S-mRNA instead of a ratio of 40-60.

Also, kass1 should not be too smaller than kdis1, or their ratio will be much larger than 40-60.

Finally we set down that kass1 = kdis1.

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When inhibiting the expression of HUB1, there is still a background splicing of 5’S site, so we need another parameter b in S-splicing.

Background S-splicing parameter b is mostly related to the ratio of spliceosome and Hub1p-modified spliceosome (Hub1_spliceosome) at equilibrium state. With higher b, L-mRNA and S-mRNA will come closer at equilibrium stat while it influence no-Hub1p situation more than Hub1p situation.

Sequence and Features