The Rna Interference ( Rnai ) Pathway

The company ‘Invivogen’ made a transfectable and simple-to-use plasmid, called psiRNA™, that allows the production of siRNAs within the cells. To transfect means to introduce foreign material into eukaryotic cells. psiRNA is an RNA polymerase III-based plasmid that produces ‘short hairpin’ RNAs (shRNAs). shRNAs are more stable than synthetic siRNAs and since they are continuously expressed within the cells rather than having to be injected into the cytoplasm, this method permits long-lasting silencing of the BLG gene.

When scientists know what gene they want to manipulate they 'introduce double stranded DNA, or dsRNA to the cell.' The dsRNA produce small double-stranded interfering RNAs, or siRNA into the cytoplasm of the cell.5 from this the expression of the gene decreases drastically but does not get entirely eliminated, therefore showing the role of the targeted gene.

With the use of CRISPR, a specific gene in the genome of a cell can be targeted and mutated to rid of the preexisting mutation. The technique works through the use of an enzyme called Cas9, which acts as the “scissors” to cut two strands of DNA at a specific location in the genome to allow for pieces of DNA to be added or removed. Another molecule of use in the process is a piece of RNA called guide RNA (gRNA). Guide RNA comprises of a small piece of pre-designed RNA sequence located within a longer RNA strand. This longer RNA strand binds to DNA and the pre-designed sequence guides the Cas9 to the correct part of the genome. This occurs for the assurance that the Cas9 enzyme cuts the right part of the genome out. The article provides specific cancers and genetic diseases and the targets for CRISPR/Cas9 that act on these mutations. Amongst the cancers, lung, thyroid, and breast cancer was mentioned. The genetic diseases mentioned were Huntington disease, Alzheimer’s and muscular

These small dsRNAs are the molecules loaded into the RISC to guide the complex to the target genes. The tiny dsRNAs act as escorts by using Watson-Crick base-pairing. Ultimately, the dicer enzyme facilitates the activation of the RNA-induced silencing complex. It belongs to the RNase III endo-ribonuclease enzyme family because it is a helicase with an RNase motif. In this experiment we tested to see if making a mutation in the dicer enzyme would inhibit its ability to cleave large dsRNAs into smaller ones. If the mutation does inhibit, it will cause the RNA interference pathway to come to a halt. Without RNA interference, the human body could stop producing oncogenes or tumor repressors. This could eventually result in

Long dsRNA s bind to Dicer to cut into smaller nucleotide fragments/segments. siRNA unwinds when it is catalysed by Argonaut. One strand remained with the Argonaut is the guide strand. The RNA guide strand with Argonaut and other proteins are combined into RNA induced silencing complex (RISC) and the other passenger RNA strand is released into nucleotides.

Since shRNA does not interact with the Drosha/DGCR8 microprocessor complex, this RNA must be designed with care in order to achieve correct downstream processing and gene suppression.1 Proper dosage of shRNAs is another factor to consider, as it has been found that they may saturate the endogenous pathway and disrupt natural miRNAs.4

RNA stands for ‘ribonucleic acid’ and it is present in all living cells. RNA’s main job is to act as a messenger carrying instructions from DNA for controlling the synthesis of proteins. RNA is similar in structure and properties to DNA. RNA, however, has only a single strand of bases. Also, rather than having the base thymine, RNA has the base uracil. Anyways, the guide RNA is composed of a miniscule pre-designed RNA sequence. This sequence is roughly 20 bases long and is located within a longer RNA framework. The scaffold part of the RNA binds to DNA and the pre-designed sequence. It ‘guides’ Cas9 to the right part of the genome. This makes sure that the Cas9 enzyme cuts at the right point in the genome.

It is greatly involved in immunity. Mostly in plants, it is greatly involved in the immune response system to prevent viruses and harmful genetic material from damaging the cell. In animals, the RNAi pathway serves an antiviral purpose, providing protection against pathogens, bacteria, and other harmful organisms that could potentially put the cell in danger. Jaronczyk’s findings exaplain that RNAi pathways also play a large role in the regulation of development of cell growth and development. It regulates the timing of morphogenesis, a process which organizes the special distribution of cells during its embryotic development stages. It also regulates the maintenance of undifferentiated cell types, or those who are not yet specialized. Finally, RNAi pathways help in RNA activation, and event in which specific short dsRNA molecules bring about the targeted gene expression. Even evidence of RNA interference pathways are relevant in the everyday lives of humans such as insecticides, genetically engineered foods, and new treatments for cancer as described by Hannon in RNAi: A Guide to Gene Silencing

transfer RNA or tRNA, has a complementary triplet of nucleotides to the mRNA codon attached

Sir2 and Hst1 have high sequence similarity (see figure 2), and in high copy numbers, they may partially compensate for one another; however, they do not function in place of one another under normal cellular conditions. The subtle differences between these two proteins therefore must determine which cofactors they interact with, and therefore what role in silencing or repression they play. In order to study this, the Vershon lab created a series of “chimera” proteins which swapped similar regions of Hst1 into Sir2 and vice versa in order to study which regions of the proteins were responsible for cofactor interactions (Mead et al, 2007). Through this study, they determined that the N-terminus of Sir2 is responsible for interaction with Sir3 and Sir4. They also determined the region of Hst1 required for interaction with Sum1 and Rfm1; this region is highly conserved, so they then created a series of individual amino acid swaps within that region to determine which amino acids were responsible for the cofactor interactions. They then

The reduction can occur through genetic modification or treatment with a reagent such as short DNA or RNA oligonucleotide that has a sequence complementary to either gene or an mRNA transcript. Gene knockdown or RNA interference (RNAi) is a natural process which cells use to turn down, or silence the activity of specific genes. It is a biological process where RNA molecules inhibit gene expression, which is caused by the destruction of specific mRNA molecules. RNAi destroys the molecular messengers that carry information, which is coded in genes to the cell’s protein factories. The molecular messengers that are being destroyed are called messenger RNAs (mRNAs) which are destroyed by the RNAi molecules as they bind to the mRNA and deactivate the mRNA, so stop it from producing polypeptide, these mRNAs are used to carry out a critical function, without this a gene is essentially inactive. The double-stranded RNA molecules that trigger RNAi get cut into small fragments by the enzyme called ‘Dicer’. These small fragments serve as guides which lead the cells RNAi machinery to mRNAs that match the genetic sequence of the fragments. After this, the machinery then slices these cellular mRNAs, which effectively destroy their messages and shut off the corresponding gene. RNAi exists in many living things, including single-celled organisms, plants and

Gene are sections of DNA that contain the instructions for making proteins. Each gene is responsible for producing a corresponding protein in a two-step process as I stated before and In gene knockdown the translation step is been interfered. Instead of directly editing DNA or inhibiting the transcription process (Gene Knockout), the key idea behind gene knockdown is intervening in gene expression prior to translation.

During the process of RNA interface long double stranded RNA is cut into small pieces by an enzyme known as “Dicer”. Those small pieces bind with proteins known as the Argonaute proteins. After this binding takes place one of the double stranded RNA becomes removed which leaves the remaining strand to bind to messenger RNA target sequences. Once they are bound, the protein can either recruit certain factors to help regulate it, destroy it, or cleave the messenger

poly-A-binding protein is a protein that not only shields the mRNA poly(A) tail, but it also interacts with sequences in the mRNA that have a general and specific role on the different mRNA metabolisms (Goss).

The process of gene knockdown has been largely unsuccessful so the scientists from AgResearch attempted the process of gene knockdown by RNA interference (RNAi). The role of the RNAi in the nucleus is in the formation of the chromatin domains that help to silence gene expression. First of all the scientists had to identify the gene/s responsible for producing the BLG protein and recognise the base sequence so that the messenger RNA (mRNA) sequence was known. This is done by the restriction enzymes, which cut the DNA at specific places so that the polymerase chain technique can be used to produce many identical copies of the nucleotide sequence. This is done by the Taq polymerase DNA to enlarge the short fragments of DNA. Gene knockdown is a technique where the expression of an organism’s genes is reduced. Gene knockdown by RNA interference (RNAi) silences the genes by mRNA degradation by inserting small interfering RNA (siRNA) into the cytoplasm. Protein-coding genes inside