Like, if I want to find out all miRNA sequences for beta-actin mRNA target, what can I do?
Thanks.

MBer:

Check out the miRBase database provided by Sanger Institute.

Here is an overview of the applications on this database:

miRBase is the new home for microRNA data, incorporating the database and gene naming roles previously provided by the miRNA Registry, and including the new miRBase Target database.

miRBase contains 3 main sections:

miRBase Sequences contains all published miRNA sequences, genomic locations and associated annotation.

miRBase Targets is a newly developed database of predicted miRNA target genes.

miRBase Registry provides a confidential service assigning official names for novel miRNA genes prior to publication of their discovery.

Here is a link to Technote from Ambion that may be helpful

Precursor miRNAs for Successful miRNA Functional Studies

Introduction:

miRNA target site identification and validation
Screening for miRNAs that regulate target gene expression
Screening for miRNAs that affect a cellular process

MicroRNAs (miRNAs) are small, regulatory RNAs that are expressed in animals and plants and affect the translation or stability of target mRNAs. The 17-24 nt, single-stranded (ss) miRNAs are derived from longer, primary transcripts termed "pri-miRNAs" [1]. The pri-miRNAs, which can be more than 1000 nt in length, contain an RNA hairpin in which one of the two strands includes the mature miRNA [1]. The hairpin, which typically comprises 60-120 nt, is cleaved from the pri-miRNA in the nucleus by the double-strand-specific ribonuclease, Drosha [1]. The resulting precursor miRNA, or "pre-miRNA," is transported to the cytoplasm via a process that involves Exportin-5 [2-4]. The pre-miRNA is further cleaved by Dicer [5] to generate a short, partially double-stranded (ds) RNA in which one strand is the mature miRNA. The mature miRNA is taken up by a protein complex that is similar, if not identical, to the RNA Induced Silencing Complex (RISC) that supports RNA interference (RNAi) [6], and miRNA-bound complex functions to regulate translation.

miRNA functional analyses can be carried out using tools that are similar to those used to analyze protein encoding genes. Up-regulation of the miRNAs can help to identify gain-of-function phenotypes while down-regulation or inhibition studies can help to identify loss-of-function phenotypes. The combination of up- and down-regulation can be used to identify not only genes that are regulated by miRNAs but also cellular processes that are affected by specific miRNAs. Two recent publications have described antisense molecules that can inhibit the activities of miRNAs [7, 8].


References (from Ambion Technotes 12(1)

1. Lee Y, Jeon K, Lee JT, Kim S, Kim VN (2002) MicroRNA maturation: stepwise processing and subcellular localization. EMBO J 21:4663-4670.

2. Lund E, Guttinger S, Calado A, Dahlberg JE, Kutay U (2004) Nuclear Export of miRNA Precursors. Science 303:95-98.

3. Yi R, Qin Y, Macara IG, Cullen BR (2003) Exportin-5 mediates the nuclear export of pre-microRNAs and short hairpin RNAs. Genes Dev 17:3011-3016.

4. Zeng Y, Cullen BR (2004) Structural requirements for pre-microRNA binding and nuclear export by Exportin 5. Nucleic Acids Res 32(16):4776-4785.

5. Lee Y, Ahn C, Han J, Choi H, Kim J, Yim J, Lee J, Provost P, Radmark O, Kim S, Kim VN (2003) The nuclear RNase III Drosha initiates microRNA processing. Nature 425:415-419.

6. Hutvagner G, Zamore PD (2002) A microRNA in a multiple-turnover RNAi enzyme complex. Science 297:2056-2060.

7. Meister G, Landthaler M, Dorsett Y, Tuschl T (2004) Sequence-specific inhibition of microRNA- and siRNA-induced RNA silencing. RNA 10(3):544-550.

8. Hutvagner G, Simard MJ, Mello CC, Zamore PD (2004) Sequence-specific inhibition of small RNA function. PLoS Biol 2(4):E98.

Also, please check out this free online resource - PicTar

PicTar is an algorithm for the identification of microRNA targets. This searchable website provides details (3' UTR alignments with predicted sites, links to various public databases etc) regarding:

(1) microRNA target predictions in vertebrates (Krek et al, Nature Genetics 37:495-500 (2005))

(2) microRNA target predictions in seven Drosophila species (Grün et al, PLoS Comp. Biol. 1:e13 (2005))

(3) microRNA targets in three nematode species (Lall et al, Current Biology 16, 1-12 (2006))

(4) human microRNA targets that are not conserved but co-expressed (i.e. the microRNA and mRNA are expressed in the same tissue) (Chen and Rajewsky, Nat Genet 38, 1452-1456 (2006)) co-expressed targets

New: co-expressed human microRNA target predictions are now available

PicTar is a project of the Rajewsky lab at NYU's Center for Comparative Functional Genomics and the Max Delbruck Centrum, Berlin

Here is a link to microInspector a scanning software for detection of miRNA binding sites.


This program was written by
Ventsislav Rusinov and Vesselin Baev

MicroInspector is run by the Tabler lab at the Institute of Molecular Biology & Biotechnology (IMBB), Heraklion,Greece.

The description of the program is accepted for publication in the 2005 web issue of
Nucleic Acid Research.

Another helpful tool is the Probabilistic miRNA Prediction tool ProMiR II

ProMiR II is an improved version of the ProMiR for more general prediction of miRNAs. ProMiR II consists of three programs: ProMiR-v, ProMiR-c, and ProMiR-g. ProMiR-v searches both conserved and non-conserved miRNAs in the vicinity of a known miRNA. ProMiR-c predicts both conserved and non-conserved miRNAs in the vicinity of a candidate (70~150 nt). ProMiR-g provides a more general service such as, the prediction of miRNA genes in a long sequence (70 nt~10 kb) of various speceis.

ProMiR II scans a long sequence or a proximal sequence by a window size (70~150 nt) and a shift size (3~10 nt) and extracts extended stem-loop sequences following the filtering parameters, such as free energy, GCratio (0.0~1.0), conservation score (0.0~1.0), and entropy (0.0~2.0) of candidate sequences. Finally, ProMiR II predicts miRNA candidates beyond a predefined threshold (0.01, 0.017, 0.033, 0.33 and 3.3) in the filtered stem-loops. ProMiR-v and ProMiR-c map the results on each species' genome with additional information, e.g. conservation score and known gene information.

Here is a link to the Introduction of ProMiR II page.

If you are working on plants this tool may be helpful also:

miRU: Plant microRNA Potential Target Finder

The program predicts plant miRNA target genes. It reports all potential sequences complementary to the query with mismatches no more than specified for each mismatch type. In addition, each mismatch is penalized according to the mismatch type and position to the miRNA. With default settings, the minimal score among all 20mers cannot exceed 3.0. This program can also be used for siRNA specificity detection. For more information about the prediction algorithm and questions about the search result, please click here.

All in-silico methods just give you predicted miRNA target sites. Some papers (e.g. Didiano & Hobert, 2006) recently show that these target sites are of limited utility as post-transcriptional gene silencing via miRNAs is likely context-dependent (e.g. one miRNA will only work if another miRNA is also present). One database that is attempting to catalogue only the experimentally-validated miRNA-target interactions is TarBase. Depending on what you want this list for, it might be worthwhile trying that database for a more high-quality/stringent result.

Ryan

MBer said:

Like, if I want to find out all miRNA sequences for beta-actin mRNA target, what can I do? Thanks.

I was also interested in exactly the same topic after hearing a seminar by Prof. Croce of OSU. I went through each of the programs suggested above but all of them seem to give different results ! So I read up some publications and this is what I found. The best way to find if your gene of interest is targeted by miRNA or targets miRNA clusters is to do a microarray using cell lines that over-express your gene of interest. Thats the good news. Affymetrix does not have any gene chips that they recommend for miRNA studies, though there are a bunch of others already on the market.