LectureChapter 11 - Gene regulation at the RNA levelChapter 12 - Processing of RNA

Today we finished chapter 11 by discussing how micro RNA (miRNA) is used to block translation of mRNA, how transcriptional attenuation (premature termination of transcription) is prevented by the action of RNA binding proteins, and how riboswitches can act to prematurely terminate transcription or inhibit translation.

In chapter 12, we talked about the kinds of RNA that can be found in prokaryotes and eukaryotes, and what are their relative quantities, compared to other organic materials in the cell. Then we had an overview of how different kinds of RNA (tRNA, rRNA, mRNA) are processed.

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Module 4, Lab 14 - BioinformaticsA simple bioinformatics pipeline to infer a phylogeny

A 'good' electropherogram of the RRss gene from an earthworm (Lumbricus; top),
and a 'bad' electropherogram of RRss gene of a cnidarian (Hydra; bottom)
(click pic for a full size view)
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During this lab students followed a simple bioinformatics pipeline (workflow) to process data similar those generated by them in the lab. We started by visualizing DNA sequencing electropherograms and analyzing the differences between a 'good' one (from which reliable information can be extracted) and a 'bad' one (from which no reliable information can be obtained).

Then we did a crude editing of the information contained in them and exported the data in fasta file format. Once we pooled sequence data (Ribonucleotide reductase small subunit [RRss], a nuclear gene) from 9 animals in a single fasta file we did a multiple sequence alignment using an on-line version of ClustalW available on the European Bioinformatics Institute website.

The alignment was used to generate a nexus file which can be used for performing phylogenetic analyses. We ran two, very basic analyses, one under the criterion of maximum parsimony (MP) and one under the criterion of maximum likelihood (ML), using an online version of PHYLIP, available at the Mobyle@Pasteur web portal. Students will compare the outcome of both analyses and write a short essay about the process followed to obtain the phylogenies.

The following learning outcomes should have been met:

• Introduction to the concept and the field of bioinformatics
• Introduction to the main sequence data repository in the Americas and one of the main in the world: NCBI
• Introduction to the main database in the NCBI website: GenBank
• Understanding of how to interpret an electropherogram (for DNA sequencing)
• Basic understanding of the fasta and nexus file formats
• Basic use of the BLAST algorithm
• Introduction to the concept of sequence alignment
• Basic understanding of how to perform a phylogenetic analysis

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Module 4, Lab 13 - BioinformaticsUsing the BLAST

DNA sequence alignment viewed with the multiple alignment editor Se-Al. On the far right there are insertions and/or deletions in several sequences (click pic for full-size image)

We a small and crude (quick and dirty?) overview of some of the few tools available to work in bioinformatics.

The main focus of our exercise was the use of the algorithm known as BLAST (Basic Local Alignment Search Tool), available at the NCBI webpage. After an overview of some of the main features of the site, we "BLASTed" (used BLAST) a few unknown protein (using 'protein BLAST') and nucleotide (using 'nucleotide BLAST') sequences. We found out what the sequences were and to what organisms they belonged. This is one of many possible uses of the BLAST algorithm.

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Chapter 11 - Regulation at the RNA level

Following with mechanisms of gene regulation at the RNA level, we discussed how translation can be repressed or activated by regulatory proteins and how can it be regulated via anti-sense RNA or alterations to ribosomes (we used phosphorilation of proteins in the ribosomal small subunit as an example).

We also discussed the role of RNA interference (RNAi) as a defense mechanism of eukaryotic cells against some viral infections, as a cell's mechanism for gene silencing, and as a research tool to study gene function by knocking-out genes.

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Module 4, Labs 13 and 14 - Bionformatics

The following links will provide fast access to the web pages you need to do your bionformatics exercises:

• National Center for Biotechnology Information (NCBI)
• DNA Data Bank of Japan (DDBJ)
• European Bioinformatics Institute (EBI)

• GenBank statistics
• Clustal W
• Phylogeny software

• NCBI's Coffee Break

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