Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2012 Nov 1;26(21):2392-407.
doi: 10.1101/gad.204438.112. Epub 2012 Oct 16.

A Triple Helix Stabilizes the 3' Ends of Long Noncoding RNAs That Lack poly(A) Tails

Affiliations
Free PMC article

A Triple Helix Stabilizes the 3' Ends of Long Noncoding RNAs That Lack poly(A) Tails

Jeremy E Wilusz et al. Genes Dev. .
Free PMC article

Abstract

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.

Figures

Figure 1.
Figure 1.
The 3′ end of MALAT1 is highly conserved and cleaved by RNase P. (A) Although there is a polyadenylation signal at the 3′ end of the MALAT1 locus, MALAT1 is primarily processed via an upstream cleavage mechanism that is mediated by the tRNA biogenesis machinery. RNase P cleavage simultaneously generates the mature 3′ end of MALAT1 and the 5′ end of mascRNA. The tRNA-like small RNA is subsequently cleaved by RNase Z and subjected to CCA addition. (B) Immediately upstream of the MALAT1 RNase P cleavage site (denoted by an arrow) is a highly evolutionarily conserved A-rich tract. Further upstream are two nearly perfectly conserved U-rich motifs separated by a predicted stem–loop structure. (C) Similar motifs are present upstream of the MEN β RNase P cleavage site. (D) The CMV-cGFP-mMALAT1_3′ sense expression plasmid was generated by placing nucleotides 6581–6754 of mouse MALAT1 downstream from the cGFP ORF. No polyadenylation signal is present at the 3′ end. (E) After transfecting the plasmids into HeLa cells, Northern blots were performed to detect expression of mascRNA and cGFP-MALAT1_3′ RNA. To verify that the 3′ end of cGFP-MALAT1_3′ RNA was accurately generated and that no additional nucleotides were added post-transcriptionally, RNase H digestion was performed prior to Northern blot analysis.
Figure 2.
Figure 2.
The U-rich motifs inhibit uridylation and degradation of the 3′ end of MALAT1. (A) Schematics of cGFP expression plasmids used in this study. (Middle) To generate a cGFP transcript ending in a canonical poly(A) tail, the mMALAT1_3′ region was replaced with either the bovine growth hormone (bGH) or the SV40 polyadenylation signal. (Bottom) To generate a nuclear-retained cGFP transcript, nucleotides 1676–3598 of mMALAT1 was placed upstream of cGFP. (B) Transfected HeLa cells were fractionated to isolate nuclear and cytoplasmic total RNA, which was then subjected to Northern blot analysis with a probe to the cGFP ORF. A probe to endogenous MALAT1 was used as a control for fractionation efficiency. (C) The SpeckleF2-MALAT1_3′ transcript was efficiently retained in the nucleus. (D) Mutations or deletions (denoted in red) were introduced into the mMALAT1_3′ region of the CMV-cGFP-mMALAT1_3′ expression plasmid. (E) The wild-type (WT) or mutant plasmids were transfected into HeLa cells, and Northern blots were performed. RNase H treatment was performed prior to the Northern blot that detected cGFP-MALAT1_3′ RNA. (F) A ligation-mediated 3′ RACE approach was used to examine the 3′ ends of cGFP-MALAT1_3′ transcripts undergoing degradation. Nucleotides added post-transcriptionally are in red. (G) RNase H treatment followed by Northern blotting was used to show that the cGFP-MALAT1_3′ Comp.14 transcript is stable. As 51 nt were deleted to generate the Comp.14 transcript, a band of only 139 nt is expected.
Figure 3.
Figure 3.
Base-pairing between U-rich motif 2 and the A-rich tract is necessary but not sufficient for MALAT1 stability. (A) Predicted secondary structure of the 3′ end of the mature Comp.14 transcript. Denoted in purple are base pairs between U-rich motif 2 and the A-rich tract that were mutated in CE. (B) Mutations (denoted in red) were introduced into the CMV-cGFP-mMALAT1_3′ expression plasmid. The full 174-nt mMALAT1_3′ region was present in these plasmids, although only the region between U-rich motif 2 and the A-rich tract is shown. (C–E) The wild-type (WT) or mutant plasmids were transfected into HeLa cells, and Northern blots were performed. RNase H treatment was performed prior to the Northern blots detecting cGFP-MALAT1_3′ RNA.
Figure 4.
Figure 4.
A triple helix forms at the 3′ end of MALAT1. (A) Base triples (denoted by dashed lines) form at the 3′ end of the mature Comp.14 transcript. This structure is similar to that shown in Figure 3A except that the orientation of the conserved stem–loop has been rotated by 90°. The U-A•U base triples that were mutated in E are denoted in purple. (B) U-A•U and C-G•C base triples form via Hoogsteen hydrogen bonds to the major grove of a Watson-Crick base-paired helix. (C) Rosetta model of the MALAT1 Comp.14 3′ end in cartoon representation. Bases 1–5 are not included to achieve modeling convergence. As in A, U-rich motif 1 is in green, U-rich motif 2 is in red, and the A-rich tract is in blue. Remaining bases are in gray. (D) Close-up view of the triple helix surrounding the nonbonded base C-11 (numbering as in A). Bases are shown in stick representation with Watson-Crick hydrogen bonds in black and Hoogsteen hydrogen bonds in red. (E) Four of the U-A•U base triples were progressively converted to C-G•C base triples in the CMV-cGFP-mMALAT1_3′ expression plasmid. In the name of each construct, the asterisk represents the Hoogsteen hydrogen bonds. The wild-type (WT) or mutant plasmids were then transfected into HeLa cells, and Western blots were performed to detect cGFP protein expression. Vinculin was used as a loading control. (F) Mutations (denoted in red) were introduced into the CMV-cGFP-mMALAT1_3′ expression plasmid. The full 174-nt mMALAT1_3′ region was present in these plasmids, although only the region around U-rich motif 1 is shown. Note that the 5′ end of each transcript is on the right side to allow a direct comparison with the structure in A. The wild-type (WT) or mutant plasmids were then transfected into HeLa cells, and Northern blots were performed.
Figure 5.
Figure 5.
The MALAT1 triple helix functions as a translational enhancer element. (A) Plasmids expressing cGFP transcripts ending in the designated 3′ end sequences were transfected into HeLa cells. The mMALAT1_3′ region and the polyadenylation signals were inserted in either the sense or antisense direction as denoted. Western blots were performed to detect cGFP protein expression. Vinculin was used as a loading control. (B) Schematic of the two-color fluorescent reporter expression system. (C) The two-color expression plasmids were transiently transfected into HeLa cells, and flow cytometry was used to measure mCherry and eYFP protein expression in single cells. Shown are box plots of the ratios of mCherry to eYFP protein expression measured in individual transfected cells ([horizontal line] median; [box] 25th–75th percentile; [error bars] 1.5× interquartile range) from a representative experiment (n = 3). (D) qPCR was used to measure the ratio of mCherry mRNA to eYFP mRNA in populations of cells transfected with the two-color expression plasmids. The data were normalized to the polyadenylated construct and are shown as mean and standard deviation values of three independent experiments. (E) Mutations or deletions (denoted in red) were introduced into the mMALAT1_3′ region of the CMV-cGFP-mMALAT1_3′ expression plasmid. (F) The wild-type (WT) or mutant plasmids were then transfected into HeLa cells, and Northern blots were performed. RNase H treatment was performed prior to the Northern blot that detects cGFP-MALAT1_3′ RNA. (G) Western blotting was used to detect cGFP expression in the transfected HeLa cells. (H) Transfected HeLa cells were fractionated to isolate nuclear and cytoplasmic total RNA, which was then subjected to Northern blot analysis. (I) Nucleotides that function in promoting translation (denoted in purple) flank the triple-helical region at the 3′ end of MALAT1.
Figure 6.
Figure 6.
Ribosome footprints are observed near the 5′ end of MALAT1 in mouse ES cells. The RNA sequencing (RNA-seq) and ribosome footprint profiles of MALAT1 in mouse embryoid bodies and mouse ES cells (grown in the presence or absence of leukemia inhibitory factor [LIF]) as determined by Ingolia et al. 2011 are shown. The MALAT1 transcription start site is denoted by an arrow on the right side of the figure.
Figure 7.
Figure 7.
A transcript ending in the MALAT1 triple helix is efficiently repressed by microRNAs in vivo. (A) Inserted into the 3′ UTR of mCherry was either a sequence perfectly complementary to let-7 or two bulged let-7-binding sites. The let-7 microRNA sequence is shown in blue. (B) HeLa cells were transfected with two-color fluorescent reporter plasmids ending in either the SV40 polyadenylation signal or the mMALAT1_3′ region with or without (denoted 0x) microRNA-binding sites. In addition, 40 nM control siRNA or exogenous let-7 microRNA was cotransfected as indicated. Flow cytometry was then used to measure mCherry and eYFP protein levels. Relative fold repression was calculated as the ratio of the mean mCherry to the mean eYFP signal of the targeted construct normalized to the equivalent ratio for the nontargeted (0x) reporter. Data are shown as mean and standard deviation values of three independent experiments. (C) qPCR was used to measure mCherry and eYFP transcript levels across the population of cells, and the relative fold repression of mCherry RNA expression was calculated analogously to above. Data are shown as mean and standard deviation values of three independent experiments.

Similar articles

See all similar articles

Cited by 142 articles

See all "Cited by" articles

Publication types

LinkOut - more resources

Feedback