RNA Processing Part B: Specific Methods
Summary Students are given a figure of a northern blot from a journal article, and are asked to interpret the results, demonstrating an understanding of both the northern blot technique and RNA processing in eukaryotic cells. Concepts and content splicing of introns nuclear vs cytoplasmic location of mRNA RNA processing nucleotide probe hybridization electrophoretic separation of RNA molecules northern blot technique Thinking skills data analysis understanding of research techniques Other skills drawing labeled diagrams.
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This problem is used in an undergraduate introductory biology course, but could also be used in upper level courses, such as genetics. The problem was designed for students to solve in-class in small groups, with faculty present to provide feedback and coach as needed as described in the accompanying module.
It was part of a handout that included other problems related to the process of transcription and RNA processing.
The concept of eukaryotic RNA processing was covered in a brief, interactive lecture. Although students had been exposed to the technique of gel electrophoresis in previous problems, they had not been taught specifically about the northern blot technique. Student problem on RNA processing and northern blot technique A. Below is a representation of a northern blot Nordstrom et al.
Northern blots are used to detect the presence of specific mRNA molecules. To do a northern blot, RNA is loaded into the wells of a gel, and separated according to size by electrophoresis. The RNA is then transferred to a membrane filter in a process called blotting. The filter is incubated with a specific probe that is radioactively labeled. Draw a picture of what was just described. A piece of film like they use for an X-ray is laid on the membrane filter and then developed. The black bands correspond to where the probe bound to the RNA sequence.
In other words, the bands show the position of mRNAs containing a complementary sequence to the probe. This type of problem exposes students to a new technique in a real context, and helps them gain practice with data interpretation skills. To become an active protein, the RNA must be 1 processed into a messenger RNA by the removal of introns, 2 translocated from the nucleus to the cytoplasm, and 3 translated by the protein-synthesizing apparatus.
In some cases, the synthesized protein is not in its mature form and 4 must be posttranslationally modified to become active.
Regulation can occur at any of these steps during development. The essence of differentiation is the production of different sets of proteins in different types of cells. In bacteria, differential gene expression can be effected at the levels of transcription, translation, and protein modification. In eukaryotes, however, another possible level of regulation exists—namely, control at the level of RNA processing and transport. There are two major ways in which differential RNA processing can regulate development.
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Here, different cells can select different nuclear transcripts to be processed and sent to the cytoplasm as messenger RNA. The same pool of nuclear transcripts can thereby give rise to different populations of cytoplasmic mRNAs in different cell types Figure 5. The second mode of differential RNA processing is the splicing of the mRNA precursors into messages for different proteins by using different combinations of potential exons.
If an mRNA precursor had five potential exons, one cell might use exons 1, 2, 4, and 5; a different cell might utilize exons 1, 2, and 3; and yet another cell type might use yet another combination Figure 5. Thus, one gene can create a family of related proteins. Roles of differential RNA processing during development. B Differential splicing, whereby the same nuclear more In the late s, numerous investigators found that mRNA was not the primary transcript from the genes. This nRNA is usually many times longer than the messenger RNA because the nuclear RNA contains introns that get spliced out during the passage from nucleus to cytoplasm.
RNA Processing Part B: Volume , Specific Methods by Melvin I. Simon | | Booktopia
Originally, investigators thought that whatever RNA was transcribed in the nucleus was processed into cytoplasmic mRNA. But studies of sea urchins showed that different cell types could be transcribing the same type of nuclear RNA, but processing different subsets of this population into mRNA in different types of cells Kleene and Humphreys , Wold and her colleagues showed that sequences present in sea urchin blastula messenger RNA, but absent in gastrula and adult tissue mRNA, were nonetheless present in the nuclear RNA of the gastrula and adult tissues.
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More genes are transcribed in the nucleus than are allowed to become mRNAs in the cytoplasm. This censoring of RNA transcripts has been confirmed for specific messages by probing for the introns and exons of specific genes.
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Gagnon and his colleagues performed such an analysis on the transcripts from the CyIIIa genes of the sea urchin Strongylocentrotus purpuratus. These genes encode calcium-binding and actin proteins, respectively, that are expressed only in a particular part of the ectoderm of the sea urchin larva.
Using probes that bound to an exon which is included in the mRNA and to an intron which is not included in the mRNA , they found that these genes were being transcribed not only in the ectodermal cells, but also in the mesoderm and endoderm. Regulation of ectoderm-specific gene expression by RNA processing. Studies of differential nRNA censoring overturned the paradigm that differential gene transcription was the ultimate means of regulating embryonic differentiation. Some RNAs stay in the nucleus to function. In one interesting case, the exons go outside the nucleus to be degraded, while the introns stay and help construct the nucleolus.
The average vertebrate nRNA consists of relatively short exons averaging about bases separated by introns that are usually much longer.
Most mammalian nRNAs contain numerous exons. By splicing together different sets of exons, different cells can make different types of mRNAs, and hence, different proteins. Whether a sequence of RNA is recognized as an exon or as an intron is a crucial step in gene regulation. What is an intron in one cell's nucleus may be an exon in another cell's nucleus. Alternative nRNA splicing is based on determining which sequences can be spliced out as introns.
This can occur in several ways Figure 5. Or some cells could fail to recognize a sequence as an intron at all, retaining it within the message. Whether a spliceosome recognizes the splice sites depends on certain factors in the nucleus that can interact with those sites and compete or cooperate with the proteins that direct spliceosome formation.
Schematic diagram of alternative nRNA splicing. Exons are represented as shaded boxes, alternatively spliced exons are represented by hatched boxes, and introns are represented by broad lines.