Sunday, 21 October 2012

Paper of the week - explaining the stability of ncRNAs in the cell

3' Polyadenylation is a key mechanism whereby mRNAs are stabilised and made ready for protein translation. One of the mysteries of molecular biology of late is how long non coding RNA (lncRNA) stability is achieved given that these molecules don't have a polyadenylation signal. Wilusz et al published a paper in Genes and Development predicting that MALAT1 is protected from 3' to 5' exonuclease activity by an RNA triple helix structure. The researchers used molecular modeling to resolve that the 3' terminus is neatly tied into a triple helix and thus likely protected from degradation. This was confirmed by mutagenesis, showing that altering bases in these regions led to a reduction in transcript stability.

MALAT1 is transcribed to form a ~6.7 kb lncRNA which is abundant in the nucleus, and also producing a small tRNA like transcript which is processed into a mature 61 nt hairpin localised to the cytosol. Both transcripts are dependant on RNase P, a ribozyme which is commonly known to be involved in tRNA processing. So it seems that MALAT1 uses features of mRNA processing (such as RNA POLII) and other features from the tRNA pathway (RNAse P, RNAse Z and CCA adding enzyme).

Surprisingly, the authors show that when they placed a triple helix motif downstream of a coding sequence, that translation of the coding sequence was enhanced, as was the overall transcript stability from exonucleases. The authors also found that regardless of whether an mRNA has a classic polyA tail or a MALAT1 like triple helix tail, that microRNAs targeted both types of transcripts to a similar extent, around 3 fold repression at the protein level.

As exemplified in data presented by ENCODE researchers, our genome is rich with with non coding RNA genes which we know very little. MALAT1 is one of the most abundant of such RNAs in the cell and its over-expression has been linked to invasiveness of cancers. It's localisation to the nuclear speckles indicates a role in RNA splicing and Ribo-seq experiments have previously shown MALAT1 RNA to be partially "protected" by ribosomes. We think that MALAT1 and other lncRNAs are just as important in the cell and relevant to disease states than genes which encode regulatory proteins like p53 and NF-κB. Time will tell.

See the original paper:

Genes Dev. 2012 Oct 16. [Epub ahead of print]A triple helix stabilizes the 3' ends of long noncoding RNAs that lack poly(A) tails.Wilusz JE, Jnbaptiste CK, Lu LY, Kuhn CD, Joshua-Tor L, Sharp PA.
Koch Institute for Integrative Cancer Research, Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA;
The MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) locus is misregulated in many human cancers and produces an abundant long nuclear-retained noncoding RNA. Despite being transcribed by RNA polymerase II, the 3' end of MALAT1 is produced not by canonical cleavage/polyadenylation but instead by recognition and cleavage of a tRNA-like structure by RNase P. Mature MALAT1 thus lacks a poly(A) tail yet is expressed at a level higher than many protein-coding genes in vivo. Here we show that the 3' ends of MALAT1 and the MEN β long noncoding RNAs are protected from 3'-5' exonucleases by highly conserved triple helical structures. Surprisingly, when these structures are placed downstream from an ORF, the transcript is efficiently translated in vivo despite the lack of a poly(A) tail. The triple helix therefore also functions as a translational enhancer, and mutations in this region separate this translation activity from simple effects on RNA stability or transport. We further found that a transcript ending in a triple helix is efficiently repressed by microRNAs in vivo, arguing against a major role for the poly(A) tail in microRNA-mediated silencing. These results provide new insights into how transcripts that lack poly(A) tails are stabilized and regulated and suggest that RNA triple-helical structures likely have key regulatory functions in vivo.

EDIT: Just a follow up to this story, there is a related paper published in PNAS, here is the abstract from PubMed:

 2012 Nov 5. [Epub ahead of print]Formation of triple-helical structures by the 3'-end sequences of MALAT1 and MENβ noncoding RNAs.Brown JAValenstein MLYario TATycowski KTSteitz JA.Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06536.Stability of the long noncoding-polyadenylated nuclear (PAN) RNA from Kaposi's sarcoma-associated herpesvirus is conferred by an expression and nuclear retention element (ENE). The ENE protects PAN RNA from a rapid deadenylation-dependent decay pathway via formation of a triple helix between the U-rich internal loop of the ENE and the 3'-poly(A) tail. Because viruses borrow molecular mechanisms from their hosts, we searched highly abundant human long-noncoding RNAs and identified putative ENE-like structures in metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) and multiple endocrine neoplasia-β (MENβ) RNAs. Unlike the PAN ENE, the U-rich internal loops of both predicted cellular ENEs are interrupted by G and C nucleotides and reside upstream of genomically encoded A-rich tracts. We confirmed the ability of MALAT1 and MENβ sequences containing the predicted ENE and A-rich tract to increase the levels of an intronless β-globin reporter RNA. UV thermal denaturation profiles at different pH values support formation of a triple-helical structure composed of multiple U•A-U base triples and a single C•G-C base triple. Additional analyses of the MALAT1 ENE revealed that robust stabilization activity requires an intact triple helix, strong stems at the duplex-triplex junctions, a G-C base pair flanking the triplex to mediate potential A-minor interactions, and the 3'-terminal A of the A-rich tract to form a blunt-ended triplex lacking unpaired nucleotides at the duplex-triplex junction. These examples of triple-helical, ENE-like structures in cellular noncoding RNAs, are unique.