MicroRNAs: Genomics, Biogenesis, Mechanism, and Function

Two different methods of presenting quantitative gene expression exist: absolute and relative quantification. Absolute quantification calculates the copy number of the gene usually by relating the PCR signal to a standard curve. Relative gene expression presents the data of the gene of interest relative to some calibrator or internal control gene. A widely used method to present relative gene expression is the comparative C(T) method also referred to as the 2 (-DeltaDeltaC(T)) method. This protocol provides an overview of the comparative C(T) method for quantitative gene expression studies. Also presented here are various examples to present quantitative gene expression data using this method.

/pdf/analyzing-real-time-pcr-data-by-the-comparative-c-t-method-tnacbujy1o.pdf

Analyzing real-time PCR data by the comparative C(T) method.

http://bartellab.wi.mit.edu/publication_reprints/Bartel_Cell09.pdf

MicroRNAs: Target Recognition and Regulatory Functions

Motivation: Biological sequence data is accumulating rapidly, motivating the development of improved high-throughput methods for sequence classification.

Results: UBLAST and USEARCH are new algorithms enabling sensitive local and global search of large sequence databases at exceptionally high speeds. They are often orders of magnitude faster than BLAST in practical applications, though sensitivity to distant protein relationships is lower. UCLUST is a new clustering method that exploits USEARCH to assign sequences to clusters. UCLUST offers several advantages over the widely used program CD-HIT, including higher speed, lower memory use, improved sensitivity, clustering at lower identities and classification of much larger datasets.

Availability: Binaries are available at no charge for non-commercial use at http://www.drive5.com/usearch

Contact: [email protected]

Supplementary information:Supplementary data are available at Bioinformatics online.

/pdf/search-and-clustering-orders-of-magnitude-faster-than-blast-1c8ksdt7ke.pdf

Search and clustering orders of magnitude faster than BLAST

Pfam is a widely used database of protein families and domains. This article describes a set of major updates that we have implemented in the latest release (version 24.0). The most important change is that we now use HMMER3, the latest version of the popular profile hidden Markov model package. This software is approximately 100 times faster than HMMER2 and is more sensitive due to the routine use of the forward algorithm. The move to HMMER3 has necessitated numerous changes to Pfam that are described in detail. Pfam release 24.0 contains 11,912 families, of which a large number have been significantly updated during the past two years. Pfam is available via servers in the UK (http://pfam.sanger.ac.uk/), the USA (http://pfam.janelia.org/) and Sweden (http://pfam.sbc.su.se/).

/pdf/the-pfam-protein-families-database-3t3lguk949.pdf

The Pfam protein families database

In Drosophila melanogaster, the iab-4/iab-8 locus encodes bi-directionally transcribed microRNAs that regulate the function of flanking Hox transcription factors. We show that bi-directional transcription, temporal and spatial expression patterns and Hox regulatory function of the iab-4/iab-8 locus are conserved between fly and the beetle Tribolium castaneum. Computational predictions suggest iab-4 and iab-8 microRNAs can target common sites, and cell-culture assays confirm that iab-4 and iab-8 function overlaps on Hox target sites in both fly and beetle. However, we observe key differences in the way Hox genes are targeted. For instance, abd-A transcripts are targeted only by iab-8 in Drosophila, whereas both iab-4 and iab-8 bind to Tribolium abd-A. Our evolutionary and functional characterization of a bi-directionally transcribed microRNA establishes the iab-4/iab-8 system as a model for understanding how multiple products from sense and antisense microRNAs target common sites.

/pdf/structure-evolution-and-function-of-the-bi-directionally-54ebqur227.pdf

Structure, evolution and function of the bi-directionally transcribed iab-4/iab-8 microRNA locus in arthropods.

Non-coding RNA (ncRNA) genes produce a functional RNA product, rather than a translated protein. The range and importance of such genes is only recently apparent, with known ncRNAs participating in a wide range of structural, regulatory, and catalytic roles within the cell. Like protein-coding genes, multiple sequence alignments of families of ncRNAs tell us much about their structure and function, and enable the formulation of statistical models for the detection of related sequences. Rfam is a database of families of ncRNAs, represented by structure-annotated multiple sequence alignments and covariance models.


Keywords:

non-coding RNA;
multiple sequence alignment;
genome annotation

Annotating Non‐Coding RNAs with Rfam

The molecular machinery for incorporating selenocysteine into proteins is present in both prokaryotes and eukaryotes. Although selenocysteine insertion has been reported in animals, plants, and protozoans, known eukaryotic selenocysteine tRNA sequences and selenocysteine insertion sequences are limited to animals and plants. Here we present clear indications of the presence of selenocysteine-tRNA and a selenocysteine insertion sequence in Plasmodium falciparum. To our knowledge, this is the first report of an identification of protozoan selenocysteine insertion machinery at the sequence level.

/pdf/a-selenocysteine-trna-and-secis-element-in-plasmodium-qir8y7br0v.pdf

A selenocysteine tRNA and SECIS element in Plasmodium falciparum.

Do interstrand hydrogen bonds contribute to β-hairpin peptide stability in solution? IR analysis of peptide folding in water

MicroRNAs are small RNAs that regulate protein levels. It is commonly assumed that the expression level of a microRNA is directly correlated with its repressive activity – that is, highly expressed microRNAs will repress their target mRNAs more. Here we investigate the quantitative relationship between endogenous microRNA expression and repression for 32 mature microRNAs in Drosophila melanogaster S2 cells. In general, we find that more abundant microRNAs repress their targets to a greater degree. However, the relationship between expression and repression is nonlinear, such that a 10-fold greater microRNA concentration produces only a 10% increase in target repression. The expression/repression relationship is the same for both dominant guide microRNAs and minor mature products (so-called passenger strands/microRNA* sequences). However, we find examples of microRNAs whose cellular concentrations differ by several orders of magnitude, yet induce similar repression of target mRNAs. Likewise, microRNAs with similar expression can have very different repressive abilities. We show that the association of microRNAs with Argonaute proteins does not explain this variation in repression. The observed relationship is consistent with the limiting step in target repression being the association of the microRNA/RISC complex with the target site. These findings argue that modest changes in cellular microRNA concentration will have minor effects on repression of targets.

/pdf/target-repression-induced-by-endogenous-micrornas-large-2ie81xtkh4.pdf

Sam Griffiths-Jones

Papers

Structure, evolution and function of the bi-directionally transcribed iab-4/iab-8 microRNA locus in arthropods.

Annotating Non‐Coding RNAs with Rfam

A selenocysteine tRNA and SECIS element in Plasmodium falciparum.

Do interstrand hydrogen bonds contribute to β-hairpin peptide stability in solution? IR analysis of peptide folding in water

Target Repression Induced by Endogenous microRNAs: Large Differences, Small Effects.