Rational and Hypothesis: My hypothesis in this aim is that mutations in UPF1 inhibit NMD thereby stabilizing premature transcripts that are translated to produce truncated proteins which promote pancreatic ASC. Previous studies have demonstrated that a subset of transcripts inhibited upon UPF1 knockdown are stabilized and translated. My objectives in this aim are: a) To determine global occupancy of mutant UPF1 on RNA b) To analyze if the mutant UPF1 bound RNA fragments are translated c) To determine which of the targets are essential to promote tumorigenesis.
A. To identify direct targets of UPF1 mutants in pancreatic cancer.
One challenge facing study of UPF1 and mammalian NMD is identification of direct regulatory targets. While genome
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Briefly, cells will be treated with cyclohexamide to arrest translating ribosomes. Extracts from these cells will be treated with RNase I to degrade regions of mRNAs not protected by ribosomes. The resulting 80S monosomes, which contain a ∼30-nucleotide RPF, will be purified on sucrose gradients and then treated to release the RPFs, which are then processed for Illumina high-throughput sequencing. In parallel, poly (A)-selected mRNA from each sample was randomly fragmented, and the resulting mRNA fragments will be processed for sequencing (mRNA-Seq) using the same protocol as that used for the RPFs. In summary, these experiments will pinpoint the targets, which are translated in UPF1 mutant cells.
C. CRISPR depletion screen to identify bonafide NMD targets
In this sub aim, my goal is to identify the fraction of NMD targets that are essential for UPF1 mediated tumorigenesis. For this purpose, I will collaborate with Dr. Vidigal who has developed a rapid cloning method for generating double guide-paired library and a computational tool for selecting guides that have minimum off-targets. Based on results from Aim 1 and 2a, I will design a customized library targeting UPF1 bound RNA targets. I will infect the library at low multiplicity of infection (MOI) into HPNE KRAS UPF1 mutant cells. I will culture cells for 30 days post transduction to maximize the identification of UPF1 mutant targets
Sufu has the ability to bind to GLI1, GLI2 and GLI3 proteins. It is theorized that GLI proteins are binded to Sufu in a head to tail orientation. GL1 is solely found in the nucleus, whereas Sufu can be found expressed in both the nucleus and the cytoplasm. However when they are both expressed together, Sufu brings the GLI1 into the cytoplasm which forces the nucleus to limit its transcription activity. The cytoplasm anchoring model suggests that GLI1 is anchored to the cytoplasm with the help of Sufu. Likewise, Sufu plays a similar role in Drosophilia by halting nuclear processes. Thus, it is found that Sufu travels back and forth between the cytoplasm and nucleus in both Drosophilia and mammals. A second model of Sufu function in mammals suggests the Sufu gene holds the ability of repressing transcription of GLIs. A study found that Sufu restricts GLI transcription by enlisting the help of the mSin3A HDAC corepressor complex. Sufu was also found to increase the ability of GLI1 to bind to DNA. Therefore, simply repressing the Sufu gene is enough to activate GLI transcription, alluding to its key role in the negative regulation of the SHH signaling
Structurally, miRNAs are initially transcribed from the genome as a long primary transcript that are cleaved or undergoes sequential processing by the RNAse III endonucleases Drosha and Dicer to yield the 17-23 mature nucleotide species (Kim, 2005). Mature miRNAs enters the RNA-induced silencing complex (RISC), thus a multiprotein complex that separate the mature strand from passenger strand and assists the interaction of mRNAs with miRNA that contain complementary sequences (Bader, 2012). As a result, mRNAs targeted by miRNA loads are either degraded or silenced in order not to be translated into protein. In the genome, a given miRNA can regulate several hundred transcripts whose molecules function within various cellular pathways including cell proliferation and apoptosis. To date, more than 50% of the activities of all protein-coding genes in mammals are controlled by miRNAs and greatly contributes to cell-type specific profiles of protein expression
Transactive response DNA binding protein 43 (TDP-43) has a molecular mass of 43 kDa and a multidomain structure that is composed of 414 amino acids, which is encoded by TARDBP (Bozzo et al., 2016; Chang et al., 2012; Igaz et al., 2011; Koyama et al., 2016). TDP-43 is a highly conserved DNA binding protein located in the nucleus for gene transcription (Chang et al., Igaz et al., 2011; 2012; Koyama et al., 2016; Neumann et al., 2006; Xu et al., 2012). However, a large number of RNA binding sites have been identified for TDP-43 that allow for pre-mRNA splicing and translational regulation including its own 3’ untranslated region 3’UTR (Ayala et al., 2011; Chang et al., 2012; Koyama et al., 2016; Xu et al., 2012). Similar to SOD1, TDP-43 is
Since its discovery as a product of the alternate reading frame of the mouse Arf/Ink4a locus signals, the Arf tumor suppressor has been identified as a key sensor of hyperproliferative stimuli such as those originating from mutant Ras and c-Myc oncoproteins (Maggi 2014. Basu 2016). p19Arf and p16Ink4a are transcribed from separate and unique first exons 1β and 1α (18 kilo base pairs [kb] apart in mice and 23kb in humans) which splice into two shared exons 2 and 3 (Fig. 1). These two genes are different tumor suppressor since p19Arf uses only exons 1 and 2 while p16Ink4a uses all of the exons 1-3 for production of the protein (Quelle 1995). This locus has a very unique genomic structure not found in other mammalian genes due to the
The Northern Blot technique allows scientists to determine the molecular weight on an mRNA and to measure the relative amounts of mRNA that are present in different samples on a single membrane. The mRNA is isolated and hybridized using this technique. It also allows for the gene to express a pattern between the human system between organs, tissues, environmental stress levels, developmental stages, and infectious pathogens. It is used to view normal tissues to that of a down regulation of tumor suppressor genes in cancerous cells. It assists scientists to recognize the functions of unknown proteins. It varies with
Mutations in the NpM1 gene are often due to translocation of the locus in chromosomes and mutated NpM1 has been characterized in a range of cancers suggesting it may be a tumour suppressor and oncogene.
Isolation of DNA along with transcriptional material in the nucleus, by the nuclear envelope, from the cytoplasm and the translational material held there gives the opportunity for exact regulation (Lange et al, 2006). The nuclear envelope provides the ability for regulation of gene expression and signal transduction. With this regulation cells can, if exposed to an extracellular signal, perceive and create an intracellular response to withstand or react to such a signal, for example, by signalling cascades with the use of mitogen-activated protein kinases (MAPKs) (Brown and Sacks, 2009). A protein kinase can phosphorylate a protein at serine, threonine or tyrosine residues which can affect its position within the cell
Top3β-C666R lost 10 folds of mRNA binding capacity while Top3β-R472Q retain mRNA binding capacity comparing to Top3β-WT
At first, we analysed the expression of Pnky LncRNA in different tumor tissues by RT-PCR. Our data were shown that Pnky is transcripts in Breast, brain, prostate and colorectal cancers as shown in fig1A . The RT-PCR after 40 cycle showed that Pnky upregulated in embryonic carcinoma cell line (NCCIT), dental pulp stem cells (DPSC) and breast
Whereas ABCE1's weak intrinsic ATPase activity is strongly activated by eRF1-bound ribosomes, stimulation by 40S subunits is weak, even though they bind strongly to ABCE1 (Pisarev et al, 2010).
This type of NGS analysis is a powerful tool for transcript and variant discovery which otherwise wouldn’t possible using traditional microarray-based methods.
RNA G-quadruplexes (GQ) structures are secondary nucleic acid structures are that can act as both necessary elements of translation and as translation repressors. formed in guanine rich regions and known to play crucial role in several biochemical processes. The RNA GQ structures are known to modulate translation of several clinically significant mRNAs such as NRAS, ZIC1, BCL-2, TRf2, FGF, VEGF,
The other ORFs are translated from subgenomic RNAs (sgRNAs), see figure 1B. ORF2-4 overlap and are therefore called the triple gene block (TGB). The encoded proteins are TGB1, TGB2 and TGB3 respectively. TGB1 is a 25kDa protein with a RNA helicase motif similar to the one found in the viral replicase. TGB2/3 contain sequences for transmembrane domains and are ER-associated. TGB1 is expressed from its own 2.1kb sgRNA (sgRNA1) whereas TGB2/3 are both expressed from the same 1.4kb sgRNA (sgRNA2). This co-translation occurs through leaky scanning. All three TGB proteins are associated with cell-to-cell movement through plasmodesmata (PD) [8]. ORF5 is expressed from a separate sgRNA of 0.9kb (sgRNA3) and encodes the coat protein (CP). CP is required both for encapsidation and cell-to-cell movement.
Luciano Marraffini and Erik Sontheimer (2008) – it is known from the work carried out by these two men that it is DNA that is the target molecule, not RNA (Marraffini, L. and Sontheimer, E., 2008).
1.2 Long non-coding RNAs Although the heterogeneous group of long non-coding RNAs (lncRNAs) account with approx. 80 % for the majority of ncRNAs in the mammalian transcriptome (Kapranov 2007), miRNAs have been in the main focus of ncRNA research in the last years. However, there is recent increase in publications describing key functions of lncRNAs in central biological processes (Taft et al. 2010) and diseases.