SummaryLong mammalian introns make it challenging for the RNA processing machinery to identify exons accurately. We find that LINE-derived sequences (LINEs) contribute to this selection by recruiting dozens of RNA-binding proteins (RBPs) to introns. This includes MATR3, which promotes binding of PTBP1 to multivalent binding sites within LINEs. Both RBPs repress splicing and 3′ end processing within and around LINEs. Notably, repressive RBPs preferentially bind to evolutionarily young LINEs, which are located far from exons. These RBPs insulate the LINEs and the surrounding intronic regions from RNA processing. Upon evolutionary divergence, changes in RNA motifs within LINEs lead to gradual loss of their insulation. Hence, older LINEs are located closer to exons, are a common source of tissue-specific exons, and increasingly bind to RBPs that enhance RNA processing. Thus, LINEs are hubs for the assembly of repressive RBPs and also contribute to the evolution of new, lineage-specific transcripts in mammals.Video Abstract

New discoveries in RNA biology underscore a need for chemical tools to clarify their roles in pathophysiological mechanisms. In certain cancers, synthesis of the let-7 microRNA tumor suppressor is blocked by an RNA binding protein (RBP) Lin28, which docks onto a conserved sequence in let-7 precursor RNA molecules and prevents their maturation. Thus, the Lin28/let-7 interaction might be an attractive drug target, if not for the well-known difficulty in targeting RNA-protein interactions with drugs. Here, we describe a protein/RNA FRET assay using a GFP-Lin28 donor and a black-hole quencher (BHQ)-labeled let-7 acceptor, a fluorescent protein/quencher combination which is rarely used in screening despite favorable spectral properties. We tested 16 000 molecules and identified N-methyl-N-[3-(3-methyl[1,2,4]triazolo[4,3-b]pyridazin-6-yl)phenyl]acetamide, which blocked the Lin28/let-7 interaction, rescued let-7 processing and function in Lin28-expressing cancer cells, induced differentiation of mouse embryonic stem cells, and reduced tumor-sphere formation by 22Rv1 and Huh7 cells. A biotinylated derivative captured Lin28 from cell lysates consistent with an on-target mechanism in cells, though the compound also showed some activity against bromodomains in selectivity assays. The Lin28/let-7 axis is presently of high interest not only for its role as a bistable switch in stem-cell biology but also because of its prominent roles in numerous diseases. We anticipate that much can be learned from the use of this first reported small molecule antagonist of Lin28, including the potential of the Lin28/let-7 interaction as a new drug target for selected cancers. Furthermore, this approach to assay development may be used to identify antagonists of other RBP/RNA interactions suspected to be operative in pathophysiological mechanisms.

Cryptic exons or pseudoexons are typically activated by point mutations that create GT or AG dinucleotides of new 5' or 3' splice sites in introns, often in repetitive elements. Here we describe two cases of tetrahydrobiopterin deficiency caused by mutations improving the branch point sequence and polypyrimidine tracts of repeat-containing pseudoexons in the PTS gene. In the first case, we demonstrate a novel pathway of antisense Alu exonization, resulting from an intronic deletion that removed the poly(T)-tail of antisense AluSq. The deletion brought a favorable branch point sequence within proximity of the pseudoexon 3' splice site and removed an upstream AG dinucleotide required for the 3' splice site repression on normal alleles. New Alu exons can thus arise in the absence of poly(T)-tails that facilitated inclusion of most transposed elements in mRNAs by serving as polypyrimidine tracts, highlighting extraordinary flexibility of Alu repeats in shaping intron-exon structure. In the other case, a PTS pseudoexon was activated by an A>T substitution 9 nt upstream of its 3' splice site in a LINE-2 sequence, providing the first example of a disease-causing exonization of the most ancient interspersed repeat. These observations expand the spectrum of mutational mechanisms that introduce repetitive sequences in mature transcripts and illustrate the importance of intronic mutations in alternative splicing and phenotypic variability of hereditary disorders.

Functional microRNAs (miRNAs) are produced from both arms of their precursors (pre-miRNAs). Their abundances vary in contextdependent fashion spatiotemporarily and there is mounting evidence of regulatory interplay between them. Here, we introduce chemically synthesized pre-miRNAs (syn-pre-miRNAs) as a general class of accessible, easily transfectable mimics of premiRNAs. These are RNA hairpins, identical in sequence to natural pre-miRNAs. They differ from commercially available miRNA mimics through their complete hairpin structure, including any regulatory elements in their terminal-loop regions and their potential to introduce both strands into RISC. They are distinguished from transcribed pre-miRNAs by their terminal 5′ hydroxyl groups and their precisely defined terminal nucleotides. We demonstrate with several examples how they fully recapitulate the properties of pre-miRNAs, including their processing by Dicer into functionally active 5p-and 3p-derived mature miRNAs. We use syn-pre-miRNAs to show that miR-34a uses its 5p and 3p miRNAs in two pathways: apoptosis during TGF-β signaling, where SIRT1 and SP4 are suppressed by miR-34a-5p and miR-34a-3p, respectively; and the lipopolysaccharide (LPS)-activation of primary human monocyte-derived macrophages, where TNF (TNFα) is suppressed by miR-34a-5p indirectly and miR-34a-3p directly. Our results add to growing evidence that the use of both arms of a miRNA may be a widely used mechanism. We further suggest that syn-pre-miRNAs are ideal and affordable tools to investigate these mechanisms.