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Analyze a peer-reviewed journal article having to do with biochemical research into mRNA degradation.
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This solution is an analysis of a research article. Although it is specific to a particular paper from a scientific journal, the answer can easily be applied to similar problems. The solution includes an analysis of each section of the article (materials and methods, results, discussion) and includes a reference list. The solution is also attached in WORD format.
Independent analysis of a research article
Term Paper #1
Bousquet-Antonelli, C., C. Presutti and D. Tollervey. (2000). Identification of a regulated pathway for nuclear pre-mRNA turnover. Cell. 102: 765-775.
In the expression of a protein, there are numerous factors and controls to be considered in determining the outcome of transcript levels. Transcription of a gene depends on signals and secondary messengers promoting transcription by the RNA polymerase. After the mRNA is synthesised further modification occurs by deadenylation of the poly-A tail and capping at the end of the pre-mRNA transcript. As well, the introns within the transcript are spliced out and the mature mRNA is transported into the cytoplasm. In this paper by Bousquet-Antonelli et al. (2000), a pathway that degrades pre-mRNA transcripts prior to splicing out of the introns is investigated. This pathway suggests another means of gene regulation whereby the cell exercises control over expression of its genes.
The nuclear exosome in yeast is a complex that is composed of ten components that are essential for normal exoribonuclease activity (Mitchell et al., 1997, Allmang et al., 1999a). Rrp4p, Rrp40p, Rrp41p, Rrp42p, Rrp43p, Rrp44p, Rrp45p, Rrp46p, Mtr3p, and Csl4p are the components that make up the exosome. The yeast exosome is found in both the nucleus and in the cytoplasm. In the cytoplasm the exosome functions in mRNA turnover and in the nucleus, the exosome will process the pre-mRNA. Components of the exosome used in this study are Rrp41p, and Rrp44p. Another exosome component, Rrp6p, is specifically nuclear and was also investigated in this paper. Along with the exosome, another exoribonuclease called Rat1p was investigated. Rat1p, unlike the exosome is a exoribonuclease (Amberg et al., 1992). All these yeast components have known human counterparts and the yeast function can thus be extrapolated.
The nuclear exosome has been shown to degrade small nuclear (snRNA), small nucleolar (snoRNA), ribosomal (rRNA) and pre-RNA spacer (Allmang et al., 1999b). The existence of the above molecules affects the stability of other molecules in the cell. The snRNA function in pre-mRNA splicing, whereas rRNA processing and modification is affected by snoRNA. As a result, the end processing which results from the exosome is important in regulating the type and amount of RNA that gets into the cytoplasm and translated into proteins. However, poly A+ RNA degradation in yeast nucleus has never been shown. Burgess and Guthrie (1993) have previously shown that in yeast with mutated reporter genes are rapidly degraded. As well, yeast-splicing mutants had reduced mRNA levels without the expected unspliced pre-mRNA accumulation. Another group (Rain and Legrain, 1997) showed that transcripts with introns get recognised and committed to splicing and will remain in the nucleus regardless of splicing. If the splicing committed transcripts remain in the nucleus then degradation probably takes place in the nucleus. These findings suggest but do not prove the existence of such a degradation pathway.
The authors in this paper hope to prove the existence of such a pathway. As well, they speculate that this pathway should be regulated. Environmental cues and stresses typically cause the down-regulation of gene expression. The authors also want to show that the growth environment regulates this pathway and as result the levels of pre-mRNA are subsequently affected.
Materials and Methods
The main method of analysis in this paper was through Northern blots of RNA from various yeast cell strains. The levels of pre- and mature mRNA allowed the authors to interpret the mechanisms of degradation of this pathway. Using an oligonucleotide designed for a gene would result in two bands representing the pre and mature forms of the mRNA. The pre-mRNA would contain introns, making it longer in length and therefore the pre-mRNA appears higher on the blot. The probes or oligonucleotides used had radioactive labels allowing them to be scanned with a phosphoimager giving a densitometric plot, showing the band intensities (see Fig 2). The densitometric plots give quantifiable results that allow comparisons with other bands on the same blot.
To determine pre-mRNA and mature mRNA levels, strains of yeast with a mutation in an exosome component were constructed. Construction of the yeast strains occurred by either transformation of yeast with select plasmids or by crossing two yeast strains. As well, strains of yeast carrying the ACT1-CUP1 reporter constructs were degraded in order to analyse the purported competition between splicing and pre-mRNA degradation. These methods will be further discussed in detail.
Crossing two strains of yeast involves taking advantage of the diploid lifecycle of yeast. During the haploid stage of growth, a yeast spore (being haploid) can combine with another spore from another strain to form a zygote, resulting in a diploid. For example, in this paper, Bousquet-Antonelli et al. (2000) crossed two strains, one of, which carried a temperature sensitive splicing mutation with another strain that carried other mutations. To create the cross, each strain would be streaked onto minimal media, which causes the yeast to sporulate and enter the haploid phase. Spores from each yeast strain would cross resulting in a diploid or zygote with the desired mutations and characteristics. Although not specifically mentioned in this paper, correct crossing can be confirmed by growth on selective media, or by PCR analysis.
The other method of creating mutants employed in this study was by transforming cells. Yeast cell transformation was carried out in a similar mechanism as to that discussed in class for bacterial cell transformation. Namely, the cells were heat shocked and a cation source was added to allow efficient uptake of plasmid DNA. The ...
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