Gracjan Michlewski

Posttranscriptional Regulation of miRNAs Harboring Conserved Terminal Loops

Summary We recently found that hnRNP A1, a protein implicated in many aspects of RNA processing, acts as an auxiliary factor for the Drosha-mediated processing of a microRNA precursor, pri-miR-18a. Here, we provide the mechanism by which hnRNP A1 regulates this event. We show that hnRNP A1 binds to the loop of pri-miR-18a and induces a relaxation at the stem, creating a more favorable cleavage site for Drosha. We found that approximately 14% of all pri-miRNAs have highly conserved loops, which we predict act as landing pads for trans-acting factors influencing miRNA processing. In agreement, we show that 2′O-methyl oligonucleotides targeting conserved loops (LooptomiRs) abolish miRNA processing in vitro. Furthermore, we present evidence to support an essential role of conserved loops for pri-miRNA processing. Altogether, these data suggest the existence of auxiliary factors for the processing of specific miRNAs, revealing an additional level of complexity for the regulation of miRNA biogenesis.

Antagonistic role of hnRNP A1 and KSRP in the regulation of let-7a biogenesis

The pluripotency-promoting proteins Lin28a and Lin28b act as post-transcriptional repressors of let-7 miRNA biogenesis in undifferentiated embryonic stem cells. The levels of mature let-7a differ substantially in cells lacking Lin28 expression, indicating the existence of additional mechanism(s) of post-transcriptional regulation. Here, we present evidence supporting a role for heteronuclear ribonucleoprotein A1 (hnRNP A1) as a negative regulator of let-7a. HnRNP A1 binds the conserved terminal loop of pri-let-7a-1 and inhibits its processing by Drosha. Levels of mature let-7a negatively correlate with hnRNP A1 levels in somatic cell lines. Furthermore, hnRNP A1 depletion increased pri-let-7a-1 processing by cell extracts, whereas its ectopic expression decreased let-7a production in vivo. Finally, hnRNP A1 binding to let-7a interferes with the binding of KSRP, which is known to promote let-7a biogenesis. We propose that hnRNP A1 and KSRP have antagonistic roles in the post-transcriptional regulation of let-7a expression.

The Splicing Factor SF2/ASF Regulates Translation Initiation by Enhancing Phosphorylation of 4E-BP1

The SR protein SF2/ASF has been initially characterized as a splicing factor but has also been shown to mediate postsplicing activities such as mRNA export and translation. Here we demonstrate that SF2/ASF promotes translation initiation of bound mRNAs and that this activity requires the presence of the cytoplasmic cap-binding protein eIF4E. SF2/ASF promotes translation initiation by suppressing the activity of 4E-BP, a competitive inhibitor of cap-dependent translation. This activity is mediated by interactions of SF2/ASF with both mTOR and the phosphatase PP2A, two key regulators of 4E-BP phosphorylation. These findings suggest the model whereby SF2/ASF functions as an adaptor protein to recruit the signaling molecules responsible for regulation of cap-dependent translation of specific mRNAs. Taken together, these data suggest a novel mechanism for the activation of translation initiation of a subset of mRNAs bound by the shuttling protein SF2/ASF.

DGCR8 HITS-CLIP reveals novel functions for the Microprocessor

The Drosha–DGCR8 complex (Microprocessor) is required for microRNA (miRNA) biogenesis. DGCR8 recognizes the RNA substrate, whereas Drosha functions as the endonuclease. Using high-throughput sequencing and cross-linking immunoprecipitation (HITS-CLIP) we identified RNA targets of DGCR8 in human cells. Unexpectedly, miRNAs were not the most abundant targets. DGCR8-bound RNAs also comprised several hundred mRNAs as well as small nucleolar RNAs (snoRNAs) and long noncoding RNAs. We found that the Microprocessor controlled the abundance of several mRNAs as well as of MALAT1. By contrast, DGCR8-mediated cleavage of snoRNAs was independent of Drosha, suggesting the involvement of DGCR8 in cellular complexes with other endonucleases. Binding of DGCR8 to cassette exons is a new mechanism for regulation of the relative abundance of alternatively spliced isoforms. These data provide insights in the complex role of DGCR8 in controlling the fate of several classes of RNAs.

Editing independent effects of ADARs on the miRNA/siRNA pathways.

Adenosine deaminases acting on RNA (ADARs) are best known for altering the coding sequences of mRNA through RNA editing, as in the GluR‐B Q/R site. ADARs have also been shown to affect RNA interference (RNAi) and microRNA processing by deamination of specific adenosines to inosine. Here, we show that ADAR proteins can affect RNA processing independently of their enzymatic activity. We show that ADAR2 can modulate the processing of mir‐376a2 independently of catalytic RNA editing activity. In addition, in a Drosophila assay for RNAi deaminase‐inactive ADAR1 inhibits RNAi through the siRNA pathway. These results imply that ADAR1 and ADAR2 have biological functions as RNA‐binding proteins that extend beyond editing per se and that even genomically encoded ADARs that are catalytically inactive may have such functions.

Structural diversity of triplet repeat RNAs.

Tandem repeats of various trinucleotide motifs are present in the human transcriptome, but the functions of these regular sequences, which likely depend on the structures they form, are still poorly understood. To gain new insight into the structural and functional properties of triplet repeats in RNA, we have performed a biochemical structural analysis of the complete set of triplet repeat transcripts, each composed of a single sequence repeated 17 times. We show that these transcripts fall into four structural classes. The repeated CAA, UUG, AAG, CUU, CCU, CCA, and UAA motifs did not form any higher order structure under any analyzed conditions. The CAU, CUA, UUA, AUG, and UAG repeats are ordered according to their increasing tendency to form semistable hairpins. The repeated CGA, CGU, and all CNG motifs form fairly stable hairpins, whereas AGG and UGG repeats fold into stable G-quadruplexes. The triplet repeats that formed the most stable structures were characterized further by biophysical methods. UV-monitored structure melting revealed that CGG and CCG repeats form, respectively, the most and least stable hairpins of all CNG repeats. Circular dichroism spectra showed that the AGG and UGG repeat quadruplexes are formed by parallel RNA strands. Furthermore, we demonstrated that the different susceptibility of various triplet repeat transcripts to serum nucleases can be explained by the sequence and structural features of the tested RNAs. The results of this study provide a comprehensive structural foundation for the functional analysis of triplet repeats in transcripts.

Tissue-specific control of brain-enriched miR-7 biogenesis

MicroRNA (miRNA) biogenesis is a highly regulated process in eukaryotic cells. Several mature miRNAs exhibit a tissue-specific pattern of expression without an apparent tissue-specific pattern for their corresponding primary transcripts. This discrepancy is suggestive of post-transcriptional regulation of miRNA abundance. Here, we demonstrate that the brain-enriched expression of miR-7, which is processed from the ubiquitous hnRNP K pre-mRNA transcript, is achieved by inhibition of its biogenesis in nonbrain cells in both human and mouse systems. Using stable isotope labeling by amino acids in cell culture (SILAC) mass spectrometry combined with RNase-assisted RNA pull-down, we identified Musashi homolog 2 (MSI2) and Hu antigen R (HuR) proteins as inhibitors of miR-7 processing in nonneural cells. This is achieved through HuR-mediated binding of MSI2 to the conserved terminal loop of pri-miR-7. Footprinting and electrophoretic gel mobility shift analysis (EMSA) provide further evidence for a direct interaction between pri-miR-7-1 and the HuR/MSI2 complex, resulting in stabilization of the pri-miR-7-1 structure. We also confirmed the physiological relevance of this inhibitory mechanism in a neuronal differentiation system using human SH-SY5Y cells. Finally, we show elevated levels of miR-7 in selected tissues from MSI2 knockout (KO) mice without apparent changes in the abundance of the pri-miR-7 transcript. Altogether, our data provide the first insight into the regulation of brain-enriched miRNA processing by defined tissue-specific factors.

Regulation of pri-miRNA Processing by a Long Noncoding RNA Transcribed from an Ultraconserved Region

Noncoding RNAs (ncRNAs) control cellular programs by affecting protein-coding genes, but evidence increasingly points to their involvement in a network of ncRNA-ncRNA interactions. Here, we show that a long ncRNA, Uc.283+A, controls pri-miRNA processing. Regulation requires complementarity between the lower stem region of the pri-miR-195 transcript and an ultraconserved sequence in Uc.283+A, which prevents pri-miRNA cleavage by Drosha. Mutation of the site in either RNA molecule uncouples regulation in vivo and in vitro. We propose a model in which lower-stem strand invasion by Uc.283+A impairs microprocessor recognition and efficient pri-miRNA cropping. In addition to identifying a case of RNA-directed regulation of miRNA biogenesis, our study reveals regulatory networks involving different ncRNA classes of importance in cancer.

An Aptamer Targeting the Apical-Loop Domain Modulates pri-miRNA Processing†

MicroRNAs (miRNAs) are short noncoding RNAs that recognize complementary bases on target mRNAs, thereby triggering either inhibition of translation initiation or mRNA degradation. They have unique expression patterns and are involved in almost every important biological process, including cell proliferation, differentiation, and apoptosis. In turn, deregulation of miRNA expression patterns is a key condition in the onset and progression of tumor development. Following the synthesis of the primary transcript (primiRNA), the maturation process of miRNAs comprises several steps. First, the pri-miRNA is hydrolyzed by the microprocessor complex, consisting of Drosha/DGCR8, to release hairpin-shaped precursor RNAs (pre-miRNAs). Subsequently, the pre-miRNA is exported into the cytoplasm and further processed by the type III ribonuclease Dicer to produce mature miRNAs. Owing to the prominent role of miRNAs in regulating gene expression, considerable efforts have been made to develop selective tools that will allow the direct targeting of miRNAs affecting either their biogenesis or function. One such class of tools is represented by the so-called antagomirs, short single-stranded 2’-methoxy-modified oligonucleotides. Antagomirs recognize mature miRNAs by complementary bases, thereby preventing miRNA–mRNA association. Here, we introduce another class of nucleic acid based molecular tools to interfere with miRNA activity, namely, RNA aptamers that specifically recognize the loop domains of a pri-miRNA and modulate its processing. Aptamers are short single-stranded nucleic acids that fold into well-defined three-dimensional structures that facilitate specific target recognition. Aptamers can be isolated by an in vitro selection process, and a wide variety of target molecules, such as proteins, cells, small molecules, and nucleic acids have been already applied for aptamer identification. Especially in the latter case, the interaction between the aptamer and the target RNA has been proven to rely on fitting three-dimensional shapes, going beyond mere recognition through complementary base pairing. We sought to elucidate whether RNA aptamers could be used as an alternative nucleic acid based molecular tool to specifically interfere with the biogenesis of individual miRNAs. Here we describe the isolation and characterization of an RNA aptamer that specifically targets the pri-miRNA polycistron 17~ 18a~ 19a~ 20a~ 19b-1~ 92. We show that the aptamer binds inter alia to the apical-loop domain of primiR18a and thereby inhibits the biogenesis of all miRNAs 1719b-1 within this cluster. Our results show that aptamers can be applied as agents that modulate pri-miRNA processing and as tools for elucidating mechanisms of this process. Furthermore, the ability to modulate the maturation of miRNA by targeting the apical-loop domain supports the importance of these domains during pri-miRNA processing. To obtain aptamers that specifically target pri-miR17~ 18a~ 19a~ 20a~ 19b-1~ 92, we applied an in vitro selection scheme in which the 791 nucleotide (nt) miRNA polycistron comprising pri-miR17 ~ 18a ~ 19a~ 20a ~ 19b-1 was biotinylated at its 5’-end and immobilized on streptavidin-coated magnetic beads (Figure 1). The beads were incubated with an RNA library comprising a 25nt random region. To avoid the participation of the constant regions of the RNA library in pri-miRNA binding, we sequestered these regions by hybridization to complementary oligodeoxynucleotides (ODNs), leaving the random nucleotides free for independent folding. After removal of all unbound RNA sequences, the retained RNAs were eluted by adding EDTA. This step essentially favors the release of those molecules that require Mg ions for RNA binding over those that exclusively rely on complementary base pairing. After seven selection cycles, enhanced pri-miRNA binding was detectable, which could be further improved by five succeeding cycles of selection and amplification (Figure S1A in the Supporting Information). The RNA library obtained from selection cycle 12 was cloned and sequenced. Amongst 17 analyzed sequences, nine revealed the consensus motif I, 5’-AACACCUC, comple[*] C. E. L nse, C. S. Hopp, Dr. A. Rentmeister, Prof. M. Famulok, Prof. G. Mayer Life and Medical Sciences (LIMES), University of Bonn Gerhard-Domagk-Strasse 1, Bonn (Germany) Fax: (+49)228-734-809 E-mail: m.famulok@uni-bonn.de gmayer@uni-bonn.de

Trim25 Is an RNA-Specific Activator of Lin28a/TuT4-Mediated Uridylation

Summary RNA binding proteins have thousands of cellular RNA targets and often exhibit opposite or passive molecular functions. Lin28a is a conserved RNA binding protein involved in pluripotency and tumorigenesis that was previously shown to trigger TuT4-mediated pre-let-7 uridylation, inhibiting its processing and targeting it for degradation. Surprisingly, despite binding to other pre-microRNAs (pre-miRNAs), only pre-let-7 is efficiently uridylated by TuT4. Thus, we hypothesized the existence of substrate-specific cofactors that stimulate Lin28a-mediated pre-let-7 uridylation or restrict its functionality on non-let-7 pre-miRNAs. Through RNA pull-downs coupled with quantitative mass spectrometry, we identified the E3 ligase Trim25 as an RNA-specific cofactor for Lin28a/TuT4-mediated uridylation. We show that Trim25 binds to the conserved terminal loop (CTL) of pre-let-7 and activates TuT4, allowing for more efficient Lin28a-mediated uridylation. These findings reveal that protein-modifying enzymes, only recently shown to bind RNA, can guide the function of canonical ribonucleoprotein (RNP) complexes in cis, thereby providing an additional level of specificity.