This is an example of how to do an indepth analysis of a research article. This analysis is on mRNA degradation.
Analyze a peer-reviewed journal article having to do with biochemical research into mRNA degradation.© SolutionLibrary Inc. solutionlibary.com 9836dcf9d7 https://solutionlibrary.com/biology/research-methods-and-experimental-design/this-is-an-example-of-how-to-do-an-indepth-analysis-of-a-research-article-this-analysis-is-on-mrna-degradation-3ic
...s in this paper used a method by Gietz et al., (1995) where the yeast cells were transformed with Lithium acetate (LiAc) as a source of cations. Gietz et al. (1995) also added single stranded carrier DNA and PEG-3350 to increase the transformation efficiency. An excess of ssDNA will bind membrane receptors on the yeast cell thus preventing the plasmid from getting bound up by the membrane. The plasmids can then pass through the membrane as it is double stranded while any free carrier DNA cannot. The addition of PEG helps to deposit the carrier and plasmid DNA onto the yeast cell membrane. Bousquet-Antonelli et al. (2000) also added 6% dimethyl sulfoxide (DMSO) prior to heat shock of the yeast cells. DMSO can penetrate membranes and its use probably helps plasmids enter the yeast cell, thus increasing the transformation efficiency. To confirm transformation, the authors used appropriate primers (see Appendix) and PCR amplified the target region. The resulting product could then be run on an agarose gel to confirm the presence of one band/product. As an added measure, the authors could have the PCR product sequenced as well
Another point to mention that was not touched on in great detail in the paper is the GAL10 construct, which contained a protein A tagged fusion protein. This construct will grow in raffinose and sucrose (RS) media used in this study for growing the yeast. However, the rrp41p gene encodes an exosome component and when grown RS medium there is a significant reduction in expression of the protein. The reduced protein expression was proven by Western blot analysis of the fusion protein product but not shown in the paper. This allows the exosome to be active as Rrp41p is an essential cell component, but there will be marked accumulation of pre-mRNA.
As mentioned, to assess the competition between splicing and degradation, the ACT1-CUP1 reporter construct was used. This construct contains an intron that is typically spliced out unless there is a splicing mutation (present in plasmids pJU97 and pJU98). Primer extension analysis will reveal three types of products of differing lengths. There will be a long product corresponding to the pre-mRNA that has an intron along with the exons. An intermediate product would be the mature mRNA with the intron splice out. The third and shortest product, found in the mutant constructs, is the intron-branch point (IBP). A lariat structure forms in the mutant as the intron gets spliced out, but will stall in plasmids with a splice site mutation. The primer extension product extends from the first exon and halts at the lariat resulting in a shortened product.
To assess the contributions to pre-mRNA degradation, the authors blocked exonuclease activity. The BEL1 gene produces a snoRNA after the BEL1 intron is spliced out. If the BEL1 with the snoRNA was subjected to exoribonucleic activity from either the 3' or 5' end we would see degradation intermediates as seen in figure 1. The intronic snoRNA would stall pre-mRNA degradation resulting in accumulation of intermediates whose degradation had not been blocked.
Finally, to assess the regulation of the nuclear exosome, the authors grew up yeast strains on different media and then probed the RNA as before. The results of this experiment and all the other experiments will be discussed further in the following section.
Results and Discussion
The first experiment in this paper looked at whether pre-mRNA is occurring in the yeast cell and if it involves a component of the exosome. The total RNA in the yeast cell was electrophoresed and transferred to Hybond. The mutant strain (GAL::rrp41) has the Rrp41p component not expressed when grown in the RS medium. When probed for various RNAs we see a 5 to 8 fold accumulation of pre-mRNA's in the mutant compared to the wild-type yeast (fig 2). There is also an increase in mature mRNA levels between the wild type and mutant stains as well. This suggests that since there are increased pre-mRNA levels, then more transcripts must be getting into the cytoplasm and subsequently transcribed. As well, there are differences in RNA levels between RNA transcripts suggesting that the exosome may degrade certain transcripts more efficiently than others may.
Fig 2 - Densitometric analysis by Bousquet-Antonelli et al. (2000) of pre-mRNA and mature mRNA levels in wild type and mutant yeast strains. The levels of mRNA were standardised to PGK1 mRNA, a gene that is not spliced.
A temperature sensitive (TS) mutant, Prp2p, was used to produce more dramatic mRNA stabilisation when combined with the above partial depletion of Rrp41p. The splicing activity of the cell is inactivated when the yeast is grown at 37oC. The defect works by causing the RNA helicase to stall and not unwind the RNA molecules properly. The result is that the pre-mRNA will accumulate in splicing inactivated strains. There was a 20 to 50 fold accumulation of pre-mRNA when the prp2p mutation was combined with the rrp41 mutation. At this point, there is nothing to degrade pre-mRNA transcripts. If you look at the mature mRNA, there are some interesting results there as well. The mature mRNA levels remained low when splicing was inactivated, suggesting that the exosome or some other component degrade the transcripts. When splicing and the exosome were inactivated, the levels of mature mRNA accumulated almost up to wild-type levels with certain genes. This experiment showed that splicing is an important component in pre-mRNA degradation.
Next, the authors needed to show that the unspliced pre-mRNA are degraded in the nucleus and not in the cytoplasm. The exosome has a nuclear and a cytoplasmic component, but the authors only focused on the nuclear component. They constructed another strain that was deficient in the nonsense-mediated-decay pathway (NMD). The NMD pathway degrades intron containing transcripts in the cytoplasm ...