iRNA in yeast

iRNA is a tool currently used in molecular biology to define the function of a gene: indeed scientists block mRNA transcription of specific gene and observe the cellular response. This technique is extremely potent and specific and allows to study one protein at time. In yeast, certain strains lack the normal RNA interference machinery and have alternative enzyme variants which can be transplanted into truly deficient species. iRNA is specially useful to study budding yeast, but this model lacks the Dicer enzyme, responsible for processing the double stranded RNAs into small interfering RNAs.
Scientists from the Whitehead Institute identified many proteins that show hallmarks of Dicer- mediated cleavage; they moved these proteins into new strains in order to reconstitute the iRNA machinery and they observed the complete silencing of all gene of interest. Thus, no limitations were found to study genes and proteins involved in yeast biology. Moreover, transposons are efficiently silenced without interfering with other genes. The system is greatly specific. Based on this positive result, scientists want to apply this finding to pathogenic yeast Candida Albicans in order to better understand the behaviour of this organism and identify some pharmacological target.

Databases

Two main databases are now available: the EMBL-EBI and the NCBI for Europe and US, respectively. These two databases are connected and all the information present are available in both systems. Another database is provided by a Japanese laboratory and is online at genome.jp.

Main databases contain information about DNA sequence, two examples are EMBL datalibrary and GenBank; all other databases regarding RNA, proteins and polymorphisms or rare diseases are connected to these two ones. Databases are usually checked by operators or software: the difference between these two control systems could be observed in the redundancy because manual control is usually more systematic than these performed by software. About proteins, three secondary databases are currently used: Swiss-Prot, TrEMBL and PIR. In these websites several bioinformatic tools are available to align sequences, predict primary and secondary structure of proteins or determine the isoelectric point, all these information are important especially at the beginning of the study. Other databases like PDB or Modbase offer three-dimensional structures of proteins and prediction of three-dimensional structures, respectively. As well as PROSITE collects information about protein motifs, functional domains and so on. Last but not least, Genome.jp is preparing a new tool, KEGG pathway, useful to retrieve information about enzymes and metabolic pathway. Good work!

What is bioinformatics?

Bioinformatics is a modern scientific discipline which wants to apply mathematical model to biology in particular to cellular and molecular biology and biochemistry. Even if in the last years important projects, such as the Genome or the Proteome project, have required a large use of bioinformatic tools, a lot of work will be done to exactly correlate biological results to in silico models. Bioinformatics is useful to create statistical model to explain biological experiments and identify significant trends. Moreover, it’s possible to generate models to compare sequences of DNA, RNA and proteins to identify relevant sequences for the evolution or for biomolecule functions. These tools are usually available for free in several websites – Expasy for instance-, are user friendly and are currently used by all scientists, even without special skills in bioinformatics. By contrast, more complex processes, like creating network with data, require great competences on biology, biochemistry, mathematics and bioinformatics. For this reason, the crucial point to obtain useful results from bioinformatics is to have a tight collaboration with other professionals in order to evaluate all the results in the right way. One big challenge of bioinformatics is to try to rationalize the biological system, overcoming the unexplainable phenomena which are often encountered.

New technologies to identify gene function

After advances in DNA sequencing technology, the major task is to determine the functional role of proteins coded by these sequenced genes. Given the broad range of different functions carried out by proteins, it’s obvious that a multiplicity of techniques will be necessary, while DNA sequencing is achieved by few, easy and simply technique. A series of strategies based on generalization and systemization of genetics are emerging now as important tool to fill the gap between sequence and activity. One of these approaches is the analysis of the effect of perturbations of gene expression, by deletion, mutation or over-expression: after one of these modifications, we expect to observe a phenotypic change.

The challenge is to quantitatively measure phenotypes with enough accuracy and depth to define gene function. Two complementary approaches for determining complex phenotypes are currently used: in the first one many different parameters are simultaneously analysed, this is an high content screen; otherwise a single or limited number of aspects are observed, but the effect of perturbing each gene is followed in combination with a second perturbation, either another mutation or a chemical treatment. This genetic interaction profiling offers a high-resolution view of the function of each gene. Saccaromyces Cerevisiae is a model really useful for this kind of studies: a complete series of deletion strains of nonessential genes has been produced and has allowed to better understand the role of proteins important for yeast biochemistry and biology. Important results have been achieved also by using conditioning mutants that selectively grow in rich media: also in this case precious information has been retrieved. Rather than loss of function studies, methods for systematic gene over-expression have been optimized. Novel approaches in this field are interested in construction of untagged proteins in order to exclude that the presence of tag could interfere with the normal function of protein self.

The main goal of this systematic studies is to maximize the information flow, while minimally compromising the accuracy of phenotype detection. The introduction of large biomolecules into cells, such as DNA, RNA allows to directly analyse the role of one gene in the cellular life, and different kind of cells (mammals, primary cells, stem cells) can be used in this approach. Biomolecule is printed in an array onto glass slides, as done in conventional microarray. A monolayer of cells is deposited on top of the arrayed molecules and cells are transfected by taking up the material from glass. By using 96-well format plate, it’s possible to analyse the effects of a large number of biomolecules in a quantitative way. A plausible example of this method application is the effect of iRNAs on cellular proliferation: iRNA can be printed on bottom plate, cells are transfected (please note that is important to define the efficiency of transfection) and proliferation rate can be measured with normal treatment with MTT. In this way, genes important in proliferation could be identified.
Future efforts will be done to exploit a vast array of data that will emerge from large-scale genomic and proteomic projects to gain a deeper knowledge of the function of biological system.

Direct RNA sequencing

Recent paper published on Nature describes an innovative method to directly sequence RNA. mRNA is usually isolated from total cellular lysate and converted in cDNA by reverse transcriptase; then, cDNA is sequenced with protocols currently used for DNA sequencing. The use of an enzyme for retro-transcription could insert mistakes into cDNA sequence with quite high frequency: an advantage of direct RNA sequencing is to bypass this step.

How does RNA sequencing occur? Polyadenylated RNAs are bound onto solid support; DNA polymerase able to use RNA as a template binds one fluorescent nucleotide complementary to the first basis of RNA. This binding generates fluorescent signal, that is read. After reading, fluorescent probe is cleaved and the following basis on RNA can be recognized and bound to another fluorescent nucleotide. In this way, after a fixed number of cycle, all RNA molecule is sequenced. Even if this technique is innovative and extremely useful, it needs to be further improved and tested because some problems due to RNA stability could arise. Moreover, it has to be ascertained the proficiency of DNA polymerase that must perfectly work. However, we hope that this method could enter as soon as possible in clinical practice.

Microinjection of yeast

Yeast is a commonly used genetic model, budding and fission have been largely studied, but few methods to introduce DNA, protein or other compounds, have been described until now. Indeed, yeast presents the rigid cell wall that avoids injection and all techniques currently used to manipulate cells. It has been described that piezo-impact micromanipulator worked well to overcome biological barriers; thus, this tool was employed also for yeast injection with good results.

The procedure, reported in the last volume of Nature Methods, allows to introduce material during fission yeast. At the microscope it’s possible to immobilize and buckle one cell, through mechanical stress, while sorbitol in the surrounding medium avoids cell disruption. Material that has to be injected enter into the cell is released from pipette and passed through the hole into the wall. The major difference between usual microinjection technique is that the pipette doesn’t touch cell surface. Why is this method so innovative? For the first time yeast has been manipulated and now inhibitors, RNA and proteins could be introduced in. Thus, new experiments could be thought and performed, keeping in mind that yeast is really diffused, as a tool for genetic studies and recombinant protein expression and other applications could be now imaged.

Overview about the transposon – based technology

Transposition is a new approach to genome manipulation. Transposable elements are DNA segments with the peculiar capability to move about the genome. Transposon-based genetic strategies are applied for the transgenesis of somatic or germ-line cells, for insertional mutagenesis, both loss or gain of function and for non viral DNA transfer into cells in cell-based clinical applications. Transposons have been identified in all organism and have been distinguished in two classes in relation of their mechanism of action. The mobility of class I elements, also named as retro-transposons, work through RNA intermediate and encode for one nucleic binding protein and an enzyme that acts as endonuclease and retro transcriptase: endonuclease generates a nick into the target DNA and retro transcriptase starts the reverse transcription of the RNA of the transposon from nicked DNA.

Class II transposable elements are simpler than those of class I and their genome, flanked by two inverted terminal repeats, encodes for a transposase protein that allows the excision of transposon and the insertion into DNA target. In this case, the transposition process could be easily controlled by separating the transposase from the transposable DNA. Several kind of transposons are now available and are used as a vector to manipulate the genomes: the first studies were performed on C. Elegans and Drosophila and only since 1997 when has been demonstrated the re-activation of the Sleeping Beauty transposon system, has been employed also in vertebrates and mammals. Parameters that have to be considered during the choice of transposon as a vector are the size of DNA that could be moved and the integration site of preference. The capacity of moving large DNA fragments varies between the species of transposons, Sleeping Beauty transposon is inhibited by large size fragments, by contrast piggyBac transposons are more tolerant to large size without reducing their efficiency. The insertion point of transposons is non-random, but occurs in hot regions of the genome, for instance intron or transcription unit, that are characteristic of each type of transposon. The integration site preference is important for the application of transposon vectors: for example, mutagenesis screening could be more efficient by using elements that tend to land in genes, while human gene therapy protocols could require vectors showing the least preference to target gene. Transposons are used to perform recessive genetic screens in embryonic stem cells and in germ-line in vivo and to transfer DNA into stem cells and oocytes and embryos. In the next future, the transposon- based technology could be applied on germ-line transgenesis of laboratory animals and in larger species, like sheep and pig. Furthermore, gene transfer could be improved into therapeutically relevant primary cells, including stem cells, allowing the implementation of ex vivo and in vivo therapies. In conclusion, transposon approaches seem to be really promising for future advances of science.

Identification of miRNA target genes

microRNA are short RNAs that are important in gene regulation in all organism, form Drosophila to human. Indeed, in mammals they induce the RNA-induced silencing complex to target sites usually located at 3’untranslated regions (UTR) of mRNAs, determining translation repression or RNA degradation. To identify putative genes targets, computational approach is generally used, while experimental validation is more difficult.

Four algorithms, miRanda, TargetScan, RNA22 and PITA, have been developed to identify targets; comparison between these algorithms allows to reduce the number of predicted genes, usually thousand of genes. So, scientists run their search with at least two algorithms and consider only the overlapping targets. Another important factor in mRNA target search is to consider the 3’ UTR: different databases of miRNA target genes propose different 3’UTR, thus to compare the result from two algorithms is crucial to start with the same database of 3’UTR. Furthermore, one miRNA could target many genes, the identification of group of genes targeted by one miRNA could give some information about the localization and the biological function of proteins, codified by these genes. Functional profiling of miRNA target can be performed through the Gene Ontology website. miRNA is a complex but really interesting tool that cells use to tune the expression of certain proteins, specially for instance during embryonic development.

miRNomics

Recent studies demonstrate an increased importance of miRNA in regulating cell function, in particular in stem cells or during embryo development, otherwise deregulated expression of miRNA often causes diseases, such as cancer. miRNAs have been identified in viruses, plants and animals and regulate protein expression by inducing degradation of mRNA target.
miRNA are short RNA sequences that recognize from ten to hundred mRNA targets, usually identified by computational analysis. Several software based on homology search and/or other parameters (free energy of folding, length of symmetric stem, A C G U content) allow us to identify mRNA targets and recognize miRNA sequence inside the genome. In April 2009 (Curr Biol. 2009 Apr 15) Dr Hervè Seitz form the University of Massachusetts Medical School proposes a new role of messenger, target of miRNA.

He wrote: “Many computationally identified miRNA targets may actually be competitive inhibitors of miRNA function, preventing miRNAs from binding their authentic targets by sequestering them”. He proposed a new model to explain the regulation of miRNA activity, based on expression of mRNA target and not on expression of miRNA itself. Once again, it’s fundamental the synergy between biological experiments and computational analysis to obtain straightforward data and enrich our knowledge of miRNome.