Science meets simplicity: A coding independent function of mRNA and pseudogene RNA.

The paper I am going to review is a demonstration of how sound logical deduction based on knowledge of basic molecular biology translated into an biological insight that was experimentally confirmed and evidentially supported.

The paper in question is this one http://www.nature.com/nature/journal/v465/n7301/abs/nature09144.html

Now, I’ve posted about miRNA and RNA interference on this blog before, as well as about the central dogma of molecular biology. The primary function of mRNA is to specify sequences that are later translated to proteins, but given that they, being RNA molecules, can interact with RNA processing/regulating machinery, it made sense to investigate if there was a coding-independent regulatory function. Before we get started, you might want to go through the article on RNA interference https://exploreable.wordpress.com/2011/01/14/right-just-as-promised-here-comes-rna-interference/ and watch a video clip on microRNA function.

Coming to the study in question, the fundamental question was; if there are two transcripts that contain binding sites for a microRNA, can levels of one have an influence on the levels of the other? (Could one transcript compete with another transcript for binding and cleavage by miRNA?). To find out, the researchers chose two transcripts; PTENP1 (a pseudogene, which evolves from the duplicate of a gene but loses the ability to form translatable transcripts) and PTEN (which is a tumour suppressor)

Before they actually tested their hypotheses, they had to verify if they could in fact compete for miRNA binding.They did this by using computational analysis of the 3′ UTR sequences to locate possible miRNAs that could bind to them both; they then settled on miR-19b and miR-20a. They verified the prediction that these microRNAs could reduce expression of both PTEN and PTENP1 by introducing them into cells, and subsequently demonstrated a causal link by showing that inhibiting this microRNAs led to derepression (restoration of high expression).

a, Working hypothesis: PTEN is protected from miRNA binding by PTENP1. miRNAs are indicated by red, blue and green structures. 5′ and 3′ UTRs, open rectangles; open reading frames, filled rectangles. b, PTEN (top) and PTENP1 (bottom) 3′ UTRs contain a highly conserved (dark grey) followed by a poorly conserved (light grey) region. PTEN-targeting miRNA seed matches within the high homology region are conserved between PTEN and PTENP1. c, Binding of PTEN-targeting miRNAs to PTENP1. Seeds and seed matches, bold; canonical pairings, solid lines; non-canonical pairings (G·U), dotted lines. d, PTEN-targeting miR-19b and miR-20a decrease PTEN and PTENP1 mRNA abundance. e, miR-17 and miR-19 family inhibitors (Imix) derepress PTENP1 abundance (left). PTEN is used as positive control (right). IC, miRNA inhibitor negative control. d, e, mean ± s.d., n ≥ 3. *P < 0.05; **P < 0.01; ***P < 0.001.

The work discussed till here managed to establish a regulatory triad; PTEN, PTENP1 and the microRNAs in question were involved in a three way relationship.Potentially, altering the levels of PTEN or PTENP1 could result in altered expression of the other gene, with phenotypic consequences. (PTEN is a tumour suppressor, and a haploinsufficient one at that, i.e, levels determine function).

The researchers then examined if knocking down PTENP1 (which has no coding function) also resulted in increased breakdown of PTEN (which is important). They used siRNA to knock out PTENP1 and investigated if levels of PTEN changed, and they found that those levels went down.

Knocking out PTENP1 using an siRNA targeted specifically against it also results in the knockdown of PTEN expression, since that frees up miRNA that would have otherwise bound to PTENP1 to go and repress PTEN expression, the reciprocal nature of the relationship is confirmed by the siRNA knockdown of PTEN causing a reduction in PTENP1 expression. The last diagram (h) confirms loss of expression by looking for reduced protein expression. The other two look at transcription.

So far, so good, they had managed to demonstrate that PTENP1 could serve to regulate PTEN expression by acting as a decoy for the requisite miRNA. How about taking things further to see if there was pathological significance?

They wanted to investigate if this functional relationship held in human tissues as well, and using microarray expression datasets from prostate cancer samples, they confirmed that the expression of both PTEN and PTENP1 was strongly correlated, implying that the demonstrated regulatory relationship held true.

Confirmation of correlation of PTENP1 and PTEN expression levels in Prostate Cancer Samples.

Tumours evolve, and cells in them undergo a host of genomic changes; frequently picking up amplifications or mutantions of genes that enhance their survival and proliferation (Oncogenes) and frequently losing those that would hinder their growth (tumour suppressors). With this in mind, the researchers examined if PTENP1 was lost by cancer cells (if it acted as a tumour suppressor by preventing miRNA from breaking down PTEN, it would make sense for cancer cells to lose PTENP1, since that would also bring about loss of PTEN activity, which is beneficial to cancer cells) and not very surprisingly, they were able to identify a subset of colon cancers that had evolved losses of genomic regions that contain PTENP1, thus demonstrating that the regulatory relationship described experimentally held, and that PTENP1 could act as a tumour suppressor.

Through the simple invocation of logic, and some cutting edge science, they’d identified a function for pseudogenes as microRNA decoys, and uncovered one method by which cancer cells may evade the action of tumour suppressor genes. This has therapeutic implications, for in cases where miRNAs are overexpressed in cancer cells, and where those lead to loss of tumour suppression, or when pseudogenes such as PTENP1 are lost, one might contemplate putting in decoy molecules to enhance the activity of tumour suppressors.

We know that miRNA is dysregulated in cancer, we know that it is a mechanism that may contribute essentially to tumourigenesis (See http://www.nature.com/nrg/journal/v10/n10/full/nrg2634.html) and we know that the evidence seen above may validate approaches that seek to employ decoys to control the effects that said dysregulation has; the future is exciting.

That is all from me this time round.  You may want to hear the author of the most recent paper I quoted natter on about miRNA dysregulation while I contemplate what to write for the next post.

Advertisements

Leave a Reply

Fill in your details below or click an icon to log in:

WordPress.com Logo

You are commenting using your WordPress.com account. Log Out / Change )

Twitter picture

You are commenting using your Twitter account. Log Out / Change )

Facebook photo

You are commenting using your Facebook account. Log Out / Change )

Google+ photo

You are commenting using your Google+ account. Log Out / Change )

Connecting to %s