Nuclear-retained Noncoding RNA Research Articles

Endoribonucleolytic Cleavage of m6A-Containing RNAs by RNase P/MRP Complex.

N6-methyladenosine (m6A) is the most abundant internal modification in RNAs and plays regulatory roles in a variety of biological and physiological processes. Despite its important roles, the molecular mechanism underlying m6A-mediated gene regulation is poorly understood. Here, we show that m6A-containing RNAs are subject to endoribonucleolytic cleavage via YTHDF2 (m6A reader protein), HRSP12 (adaptor protein), and RNase P/MRP (endoribonucleases). We demonstrate that HRSP12 functions as an adaptor to bridge YTHDF2 and RNase P/MRP, eliciting rapid degradation of YTHDF2-bound RNAs. Transcriptome-wide analyses show that m6A RNAs that are preferentially targeted for endoribonucleolytic cleavage have an HRSP12-binding site and a RNase P/MRP-directed cleavage site upstream and downstream of the YTHDF2-binding site, respectively. We also find that a subset of m6A-containing circular RNAs associates with YTHDF2 in an HRSP12-dependent manner and is selectively downregulated by RNase P/MRP. Thus, our data expand the known functions of RNase P/MRP to endoribonucleolytic cleavage of m6A RNAs.

Targeting HOTAIR Induces Mitochondria Related Apoptosis and Inhibits Tumor Growth in Head and Neck Squamous Cell Carcinoma in vitro and in vivo.

Homeobox (HOX) transcript antisense RNA (HOTAIR), a long nuclear-retained noncoding RNA (lncRNA), is overexpressed in a variety of human cancers. Increasing evidence shows that HOTAIR plays a vital role in cancer initiation and progression by affecting cell cycle progress, apoptosis and invasion. However, whether HOTAIR serves as a target of therapeutic potential and the underlying mechanism in head and neck squamous cell carcinoma (HNSCC) is still unclear. Thus, we employed a HOTAIR specific siRNA to deplete its expression in two human HNSCC cell lines, Tca8113 and Tscca. The flow cytometry (FCM) analysis showed that HOTAIR depletion induced tumor cell apoptosis in vitro. JC-1 probe examination showed that the mitochondrial membrane potential was changed significantly by HOTAIR blockage. Mitochondrial calcium uptake 1(MICU1) dependent cell death was induced by HOTAIR depletion. Protein expression analysis indicated that mitochondrial related cell death pathway (Bcl-2, BAX, Caspase-3, Cleaved Caspase-3, Cytochrome c) involved in HOTAIR dependent apoptosis process. Moreover, a Tscca derived xenograft tumor model was employed to further validate that injection of HOTAIR siRNA inhibited tumor growth. In summary, we suggested that HOTAIR inhibition could be developed as a new therapeutic in HNSCC treatments.

A triple helix stabilizes the 3′ ends of long noncoding RNAs that lack poly(A) tails

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.

The Nuclear-Retained Noncoding RNA MALAT1 Regulates Alternative Splicing by Modulating SR Splicing Factor Phosphorylation

Alternative splicing (AS) of pre-mRNA is utilized by higher eukaryotes to achieve increased transcriptome and proteomic complexity. The serine/arginine (SR) splicing factors regulate tissue- or cell-type-specific AS in a concentration- and phosphorylation-dependent manner. However, the mechanisms that modulate the cellular levels of active SR proteins remain to be elucidated. In the present study, we provide evidence for a role for the long nuclear-retained regulatory RNA (nrRNA), MALAT1 in AS regulation. MALAT1 interacts with SR proteins and influences the distribution of these and other splicing factors in nuclear speckle domains. Depletion of MALAT1 or overexpression of an SR protein changes the AS of a similar set of endogenous pre-mRNAs. Furthermore, MALAT1 regulates cellular levels of phosphorylated forms of SR proteins. Taken together, our results suggest that MALAT1 regulates AS by modulating the levels of active SR proteins. Our results further highlight the role for an nrRNA in the regulation of gene expression.

A long nuclear-retained non-coding RNA regulates synaptogenesis by modulating gene expression

A growing number of long nuclear-retained non-coding RNAs (ncRNAs) have recently been described. However, few functions have been elucidated for these ncRNAs. Here, we have characterized the function of one such ncRNA, identified as metastasis-associated lung adenocarcinoma transcript 1 (Malat1). Malat1 RNA is expressed in numerous tissues and is highly abundant in neurons. It is enriched in nuclear speckles only when RNA polymerase II-dependent transcription is active. Knock-down studies revealed that Malat1 modulates the recruitment of SR family pre-mRNA-splicing factors to the transcription site of a transgene array. DNA microarray analysis in Malat1-depleted neuroblastoma cells indicates that Malat1 controls the expression of genes involved not only in nuclear processes, but also in synapse function. In cultured hippocampal neurons, knock-down of Malat1 decreases synaptic density, whereas its over-expression results in a cell-autonomous increase in synaptic density. Our results suggest that Malat1 regulates synapse formation by modulating the expression of genes involved in synapse formation and/or maintenance.

Altered Nuclear Retention of mRNAs Containing Inverted Repeats in Human Embryonic Stem Cells: Functional Role of a Nuclear Noncoding RNA

In many cells, mRNAs containing inverted repeats (Alu repeats in humans) in their 3' untranslated regions (3'UTRs) are inefficiently exported to the cytoplasm. Nuclear retention correlates with adenosine-to-inosine editing and is in paraspeckle-associated complexes containing the proteins p54(nrb), PSF, and PSP1 alpha. We report that robust editing activity in human embryonic stem cells (hESCs) does not lead to nuclear retention. p54(nrb), PSF, and PSP1 alpha are all expressed in hESCs, but paraspeckles are absent and only appear upon differentiation. Paraspeckle assembly and function depend on expression of a long nuclear-retained noncoding RNA, NEAT1. This RNA is not detectable in hESCs but is induced upon differentiation. Knockdown of NEAT1 in HeLa cells results both in loss of paraspeckles and in enhanced nucleocytoplasmic export of mRNAs containing inverted Alu repeats. Taken together, these results assign a biological function to a large noncoding nuclear RNA in the regulation of mRNA export.

3′ End Processing of a Long Nuclear-Retained Noncoding RNA Yields a tRNA-like Cytoplasmic RNA

MALAT1 is a long noncoding RNA known to be misregulated in many human cancers. We have identified a highly conserved small RNA of 61 nucleotides originating from the MALAT1 locus that is broadly expressed in human tissues. Although the long MALAT1 transcript localizes to nuclear speckles, the small RNA is found exclusively in the cytoplasm. RNase P cleaves the nascent MALAT1 transcript downstream of a genomically encoded poly(A)-rich tract to simultaneously generate the 3' end of the mature MALAT1 transcript and the 5' end of the small RNA. Enzymes involved in tRNA biogenesis then further process the small RNA, consistent with its adoption of a tRNA-like structure. Our findings reveal a 3' end processing mechanism by which a single gene locus can yield both a stable nuclear-retained noncoding RNA with a short poly(A) tail-like moiety and a small tRNA-like cytoplasmic RNA.