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 39 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 39 ends of MALAT1 and the MEN b long noncoding RNAs are protected from 39-59 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.

In human transcriptional regulation, DNA-sequence-specific factors can associate with intermediaries that orchestrate interactions with a diverse set of chromatin-modifying enzymes. One such intermediary is HCFC1 (also known as HCF-1). HCFC1, first identified in herpes simplex virus transcription, has a poorly defined role in cellular transcriptional regulation. We show here that, in HeLa cells, HCFC1 is observed bound to 5400 generally active CpG-island promoters. Examination of the DNA sequences underlying the HCFC1-binding sites revealed three sequence motifs associated with the binding of (1) ZNF143 and THAP11 (also known as Ronin), (2) GABP, and (3) YY1 sequence-specific transcription factors. Subsequent analysis revealed colocalization of HCFC1 with these four transcription factors at~90% of the 5400 HCFC1-bound promoters. These studies suggest that a relatively small number of transcription factors play a major role in HeLacell transcriptional regulation in association with HCFC1.[Supplemental material is available for this article.]In eukaryotes, DNA-sequence-specific transcription factors and chromatin-modifying activities work together to regulate the initiation of transcription at promoters by core-promoter-binding factors and RNA polymerases. There exists also a more limited class of transcriptional regulators whose members coordinate the interaction of the DNA-binding transcription factors and chromatin-modifying activities. One of these factors is the host-cell factor HCFC1 (also known as HCF-1), which was discovered in studies of herpes simplex virus (HSV) transcription (for reviews, see Kristie et al. 2010) and for which a mechanistic understanding of its cellular role has remained relatively enigmatic, largely because it does not display DNA-binding activity.HCFC1 is synthesized as a 2035-amino-acid precursor that is cleaved by O-GlcNAc transferase (OGT) to generate a heterodimeric complex of amino-terminal HCFC1 N and carboxy-terminal HCFC1 C subunits (Capotosti et al. 2011) that regulate different aspects of the cell-division cycle ( Julien and Herr 2003).Although HCFC1 does not display direct DNA-binding activity, it associates with chromatin via a Kelch-repeat domain within the HCFC1 N subunit (Wysocka et al. 2001). The Kelch-repeat domain is predicted to form a b-propeller structure that binds to a short sequence motif, D / E HxY, called the HCFC1-binding motif (HBM) (Freiman and Herr 1997;Lu et al. 1998), which is found in several HCFC1-associated DNA-binding transcription factors (for review, see Zargar and Tyagi 2012). HCFC1 likewise associates with a constellation of chromatin-modifying activities. These latter activities include the histone H3 lysine 4 (H3K4) methyltransferases SETD1A and mixed lineage leukemia 1 (MLL), histone demethylases KDM1A and PHF8, histone acetyltransferase (HAT) KAT8, histone deacetylase (HDAC) SIN3A, glycosyl transferase OGT, ubiquitin hydrolase RNF2 (BAP-1), and the phosphatase PPA1 (for references, see Zargar and Tyagi 2012). Both these DNAbinding transcrip...

Summary MicroRNAs (miRNAs) regulate diverse biological processes by repressing mRNAs, but their modest effects on direct targets, together with their participation in larger regulatory networks, make it challenging to delineate miRNA-mediated effects. Here, we describe an approach to characterizing miRNA-regulatory networks by systematically profiling transcriptional, post-transcriptional and epigenetic activity in a pair of isogenic murine fibroblast cell lines with and without Dicer expression. By RNA sequencing (RNA-seq) and CLIP (crosslinking followed by immunoprecipitation) sequencing (CLIP-seq), we found that most of the changes induced by global miRNA loss occur at the level of transcription. We then introduced a network modeling approach that integrated these data with epigenetic data to identify specific miRNA-regulated transcription factors that explain the impact of miRNA perturbation on gene expression. In total, we demonstrate that combining multiple genome-wide datasets spanning diverse regulatory modes enables accurate delineation of the downstream miRNA-regulated transcriptional network and establishes a model for studying similar networks in other systems.

MicroRNAs (miRNAs) are critical to proliferation, differentiation, and development. Here, we characterize gene expression in murine Dicer-null adult mesenchymal stem cell lines, a fibroblast cell type. Loss of Dicer leads to derepression of let-7 targets at levels that exceed 10-fold to 100-fold with increases in transcription. Direct and indirect targets of this miRNA belong to a mid-gestation embryonic program that encompasses known oncofetal genes as well as oncogenes not previously associated with an embryonic state. Surprisingly, this mid-gestation program represents a distinct period that occurs between the pluripotent state of the inner cell mass at embryonic day 3.5 (E3.5) and the induction of let-7 upon differentiation at E10.5. Within this mid-gestation program, we characterize the let-7 target Nr6a1, an embryonic transcriptional repressor that regulates gene expression in adult fibroblasts following miRNA loss. In total, let-7 is required for the continual suppression of embryonic gene expression in adult cells, a mechanism that may underlie its tumor-suppressive function.