'micro-RNA' - Small regulators with Global Impact

RNA interference (RNAi) is a system within living cells that helps to control which genes are active and how active they are.
---Two types of small RNA molecules – microRNA (miRNA) and small interfering RNA (siRNA) – are central to RNA interference.
---RNAs are the direct products of genes, and these small RNAs can bind to specific other RNAs and either increase or decrease their activity, for example by preventing a messenger RNA from producing a protein.
---RNA interference has an important role in defending cells against parasitic genes – viruses and transposons – but also in directing development as well as gene expression in general.

Ref:-http://www.ncbi.nlm.nih.gov/pubmed/12110901

----Small Temporal RNA or stRNA regulate gene expression during roundworm development by preventing the mRNAs they bind to being translated.
---In contrast to siRNA, stRNAs downregulate expression of target RNAs after translation initiation without affecting mRNA stability.
---Nowadays, stRNAs are better known as miRNAs.
---MicroRNAs (miRNAs) are 22-24 nucleotides in length, and downregulate gene expression by attaching themselves to messenger.
------from the structural and functional similarities it can be clearly concluded that " micro RNA refer as small temporal RNA".

Ref:- http://www.nature.com/horizon/rna/background/micrornas.html

---The population where by miRNA is being studied and talked to is of plant and animal systems in various forms, and even in viruses.
---The sequences involved in miRNA based gene expression and regulation appear to be highly conserved between individuals and indeed species, but their populations and compositions within cells correlate very strongly with specific cell or tissue types, developmental stages and/or disease states.
--profiling of these populations as biomarkers should be a very powerful diagnostic and prognostic tool in paving paths to new therapeutics in cancer.
---Recent technology for detecting and analyzing miRNAs within cells has already advanced rapidly and this has paved to give a notable successes in the profiling of metastatic cancers.
hence the statement.......

Ref:-1) http://cancerres.aacrjournals.org/cgi/content/full/67/10/4553
2) http://linkinghub.elsevier.com/retrieve/pii/S1590865808006294

1)No it is not possible to overexpress any gene if the associated regulating micro RNA is inhibited.
---just it is based on "all or none?" law -- means either it will express or not at all.

2)Yes it is possible to identify and inhibit the micro RNA that regulate the size of the friuts but currently the same is under research.
----It has been found that Differentially expressed microRNAs help during solid endosperm development in coconut

Ref:-http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6TC3-4WXRDS0-1&_user=10&_rdoc=1&_fmt=&_orig=search&_sort=d&_docanchor=&view=c&_searchStrId=1102377371&_rerunOrigin=google&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=fa2790cd01ddc1ab248aeb779543592f

----MiRNAs are key players in each of the processes. Because of their ability
to quickly alter the synthesis of specific proteins they are most suitable for
this type of regulation.
----It was indeed shown that in several cancer cells some miRNA levels are
changed and, more interestingly, when the missing miRNA is introduced to
the cancer cells they stop proliferating.
----Thie above senetence clearly shows distinct variations in miRNA expression and functional behaviour observed in normal and diseased state of cells.

Ref:- http://www.pharmaprojects.com/therapy_analysis/MICROrna-0808.html

No, Miss Komal there is no different mechanism in miRNA functioning in cardiac diseases and AIDS but their main working principle is gene regulaton in these cases too.

---First of all I would like to inform you that here the presentation is on microRNA and not mRNA.So I limit my answers to my topic only.

---"The miScript PCR System " is a very eficient system for detection of miRNA throughout plant and animal systems in various form.For details on same please follow the reference link below.

Ref:- http://www1.qiagen.com/literature/qiagennews/weeklyarticle/09_06/e13/Def...

1)Yes surface antigens such as CD3e, B220, Gr-1, Mac-1, and Ter119 are present on miRNA.

2)The main function of miRNAs appears to be in gene regulation.In animals, microRNAs more often only partially base pair and inhibit protein translation of the target mRNA(this exists in plants as well but is less common).

3)Yes its an obvious that since miRNAs appears to be involved in gene regulation, genetic mutations can affect the expression of miRNA.

4)Regarding isolation i have alreadt mentioned the details in ppt and to a comment to Dr. T.E.G.K. Murthy below..please do refer for details.

5)No recent researches have shown most of the species in general have similar type of nature and regulate similar gene regulation.

Ref:- 1) http://www.ambion.com/techlib/resources/miRNA/index.html
2) http://linkinghub.elsevier.com/retrieve/pii/S0301472X06007466
3) http://www.microrna.org/

---MicroRNAs (miRNAs) play an important role in gene regulatory networks in animals.
---Yet, the mechanistic details of their function in translation inhibition or messenger RNA (mRNA) destabilization remain controversial.
---To directly examine the earliest events in this process, an in vitro translation system was developed using mouse Krebs-2 ascites cell–free extract that exhibits an authentic miRNA response.
---It has been found that translation initiation, specifically the 5' cap recognition process, is repressed by endogenous let-7 miRNAs within the first 15 minutes of mRNA exposure to the extract when no destabilization of the transcript is observed.
---Results indicate that inhibition of translation initiation is the earliest molecular event effected by miRNAs.
---Other mechanisms, such as mRNA degradation, may subsequently consolidate mRNA silencing.

Ref:- http://www.sciencemag.org/cgi/content/abstract/317/5845/1764

"A locked nucleic acid (LNA), often referred to as inaccessible RNA, is a modified RNA nucleotide."

-----Locked Nucleic Acids (LNA™) are a class of nucleic acid analogues in which the ribose ring is “locked” by a methylene bridge connecting the 2’-O atom with the 4’-C atom.
---For the in situ detection of miRNA and expression profiling the use of LNA is an efficient method.
--- for more and detailed information about working of same please follow the links in reference.....

Ref:- 1) http://www.geneworks.com.au/library/LNA_-_Locked_Nucleic_Acid.pdf
2) http://www.exiqon.com/microrna-in-situ-Hybridization

Thanks for your comments .......
1)Relative to efforts to decipher the genome, miRNA research is still relatively new and is important because of its role in gene regulation and gene expression.
--The big tools suppliers will do well in this segment, but the broad set of existing technologies employed in miRNA research lends itself to multiple niche market participants as well.
--So still it will take time for miRNA to hit Indian markets.
2)Traditional RNA isolation methods are not well suited for isolation of small RNAs such as miRNAs.
--But Now upcomong companies such as Ambion,Exion offers kits specifically developed for isolation of small RNAs, their purification and further production...
...For eg..the mirVana™ miRNA Isolation Kit and the mirVana™ PARIS™ Kit.
---These kits have simplified the work a lot.

Ref:- 1) http://www.researchandmarkets.com/reports/1061105/microrna_research_tren...
2) http://www.ambion.com/techlib/tn/132/5.html

Thanks for your comments Sir.......

1) Recently developed miRNA isolation kits are an effective way to isolate miRNAs.
-----The mirVana miRNA Isolation Kit uses a rapid procedure to isolate microRNA (miRNA) from tissues and cells. The fast and efficient glass fiber filter (GFF) based method isolates total RNA ranging in size from kilobases down to 10mers.

2)Because of the similar chemical structures of miRNAs with DNAs, the novel delivery strategies for DNAs could be applied on miRNAs.
----These strategies include the use of polymeric carriers, micelles, nanoparticles and conjugation to a targeting ligand.
----Tumor-targeted liposomes can potentially improve the therapeutic efficacy of miRNAs by achieving sustained plasma concentrations, enhanced accumulation in tumor tissues, as well as increased rates of internalization by tumor cells.

3)No, there are no immunogenic problems associated with the administration of miRNA.

Ref.:- http://www.ambion.com/catalog/CatNum.php?AM1561
------ http://www.nsec.ohio-state.edu/briefs/miRNA.pdf

Thanks for your comments Sir.......

Studies have shown that miRNAs possess a high degree of stability.
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--An investigation of the stability of miRNAs isolated from clinical samples of B lymphocytes (chronic lymphocytic leukemia) was made by the most commonly utilized method based on a Trizol/TRI-Reagent solution (RNAs stored at −80 °C).

--To assess the stability of miRNAs, a Real Time-PCR analysis was performed for a panel of 29 miRNAs from a freshly isolated RNA sample and after 14 days storage at −80 °C.
--Furthermore, a Real Time-PCR analysis was repeatedly performed for a stored RNA sample over a period of approximately 10 months.
--It was observed high stability of isolated miRNAs and respective cDNAs.

--The reproducibility and efficiency of the Trizol/TRI-Reagent isolation method was also tested and compared to the mirVana Isolation kit (Ambion) and
RNeasy kit (Qiagen).
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-----In conclusion, Trizol/TRI-Reagent based isolation is a robust reproducible method, and obtained miRNA samples do not show any tendency to degradation when properly stored and handled.

Ref.:- http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WBK-4X8CCW5-7...

Thanks for your comments .......

A good question Mr. Patel,
-------- No, the change in genetic material may not cause any bad effect on healthy or normal cells.It is explained as below:

----It has been indeed shown that in several cancer cells some miRNA levels are changed and, more interestingly, when the missing miRNA is introduced to
the cancer cells they stop proliferating.

----So there is no such danger as in anti-sense therapy.Here we are not activating or deactivating a gene but we are introducing a missing miRNA which normal cells do have in them but cancerous ones do not..Same is for other diseases too...
naturally benefit:risk ratio is higher.

Ref:- http://scienceandreason.blogspot.com/2008/02/microrna-and-cancer.html
thank you.....

Yes, mi-RNA are proving to be very useful in targeting the cancer cells.

-----------Three important observations early in the history of miRNAs suggested a potential role in human cancer.

1)The earliest miRNAs discovered in the roundworm C. elegans and the fruit fly Drosophila were shown to control cell proliferation and apoptosis. Their deregulation may therefore contribute to proliferative diseases such as cancer.

2)When human miRNAs were discovered, it was noticed that many miRNA genes were located at fragile sites in the genome or regions that are commonly amplified or deleted in human cancer.

3)Malignant tumors and tumor cell lines were found to have widespread deregulated miRNA expression compared to normal tissues.

--In addition to protein-coding oncogenes and tumor sup-pressor genes, we will have to take into account miRNAs and their regulatory networks if we aim to understand the complex processes underlying malignant transformation and target cancer cells.

------It has been indeed shown that in several cancer cells some miRNA levels are changed and, more interestingly, when the missing miRNA is introduced to the cancer cells they stop proliferating.

Ref:- http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2151131/
thank you.........

No sir microRNAs And prions are not similar or have any connection.

"Prions"- the word itself derives from ‘proteinaceous infectious particle’; meaning that the infectious agent consists only of protein with no nucleic acid genome.

---Prions are the only known example of infectious pathogens that are devoid of nucleic acid.

----All other infectious agents, like bacteria, viruses, fungi possess genomes composed of either DNA or RNA that direct the synthesis of their progeny.

Ref :- http://medind.nic.in/jac/t03/i4/jact03i4p334.pdf

Thank you.....

Factors that regulate the functions of miRNAs are as below:

1)Transcriptional regulation factors:

Serum response factor (SRF),myocyte enhancer factor 2(Mef2) and MyoD, which are all master regulators of myogenic differentiation, are required for regulation of miR-1–1 and miR-1–2 promoter regions in vivo and in vitro.

2)Post Transcriptional regulatory factors:

The biogenesis of miRNAs, which involves initial transcription, processing and export from the nucleus,and further processing into the mature form of miRNA,
seems to be regulated at multiple steps.
For example, miR-1 and miR-133 share common cis- and trans-regulation mechanisms, but the relative abundance of miR-1 or miR-133 differs dynamically in the heart and skeletal muscle at distinct stages of development, which
reflects a higher order of processing regulation.

Additional information on same for other viewers too:-

The recent discovery that RNA-editing enzymes can alter the cleavage site in pri-miR-142 within hematopoietic cells and thereby regulate the pro-cessing of miR-142 is exciting, and might represent a mechanism that is more widely used to control miRNA activity.