Shawn W. Foley

Global Analysis of the RNA-Protein Interaction and RNA Secondary Structure Landscapes of the Arabidopsis Nucleus

Posttranscriptional regulation in eukaryotes requires cis- and trans-acting features and factors including RNA secondary structure and RNA-binding proteins (RBPs). However, a comprehensive view of the structural and RBP interaction landscape of nuclear RNAs has yet to be compiled for any organism. Here, we use our ribonuclease-mediated structure and RBP-binding site mapping approaches to globally profile these features in Arabidopsis seedling nuclei in vivo. We reveal anticorrelated patterns of secondary structure and RBP binding throughout nuclear mRNAs that demarcate sites of alternative splicing and polyadenylation. We also uncover a collection of protein-bound sequence motifs, and identify their structural contexts, co-occurrences in transcripts encoding functionally related proteins, and interactions with putative RBPs. Finally, using these motifs, we find that the chloroplast RBP CP29A also interacts with nuclear mRNAs. In total, we provide a simultaneous view of the RNA secondary structure and RBP interaction landscapes in a eukaryotic nucleus.

Minor introns are embedded molecular switches regulated by highly unstable U6atac snRNA

Eukaryotes have two types of spliceosomes, comprised of either major (U1, U2, U4, U5, U6) or minor (U11, U12, U4atac, U6atac; <1%) snRNPs. The high conservation of minor introns, typically one amidst many major introns in several hundred genes, despite their poor splicing, has been a long-standing enigma. Here, we discovered that the low abundance minor spliceosome’s catalytic snRNP, U6atac, is strikingly unstable (t½<2 hr). We show that U6atac level depends on both RNA polymerases II and III and can be rapidly increased by cell stress-activated kinase p38MAPK, which stabilizes it, enhancing mRNA expression of hundreds of minor intron-containing genes that are otherwise suppressed by limiting U6atac. Furthermore, p38MAPK-dependent U6atac modulation can control minor intron-containing tumor suppressor PTEN expression and cytokine production. We propose that minor introns are embedded molecular switches regulated by U6atac abundance, providing a novel post-transcriptional gene expression mechanism and a rationale for the minor spliceosome’s evolutionary conservation. DOI: http://dx.doi.org/10.7554/eLife.00780.001

The Conservation and Function of RNA Secondary Structure in Plants.

RNA transcripts fold into secondary structures via intricate patterns of base pairing. These secondary structures impart catalytic, ligand binding, and scaffolding functions to a wide array of RNAs, forming a critical node of biological regulation. Among their many functions, RNA structural elements modulate epigenetic marks, alter mRNA stability and translation, regulate alternative splicing, transduce signals, and scaffold large macromolecular complexes. Thus, the study of RNA secondary structure is critical to understanding the function and regulation of RNA transcripts. Here, we review the origins, form, and function of RNA secondary structure, focusing on plants. We then provide an overview of methods for probing secondary structure, from physical methods such as X-ray crystallography and nuclear magnetic resonance (NMR) imaging to chemical and nuclease probing methods. Combining these latter methods with high-throughput sequencing has enabled them to scale across whole transcriptomes, yielding tremendous new insights into the form and function of RNA secondary structure.

Transcriptome-wide measurement of plant RNA secondary structure.

RNAs fold into intricate and precise secondary structures. These structural patterns regulate multiple steps of the RNA lifecycle, while also conferring catalytic and scaffolding functions to certain transcripts. Therefore, a full understanding of RNA posttranscriptional regulation requires a comprehensive picture of secondary structure. Here, we review several high throughput sequencing-based methods to globally survey plant RNA secondary structure. These methods are more accurate than computational prediction, and more scalable than physical techniques such as crystallography. We note hurdles to reliably measuring secondary structure, including RNA-binding proteins, RNA base modifications, and intramolecular duplexes. Finally, we survey the functional knowledge that has been gleaned from each of these methods, and identify some unanswered questions that remain.

Differential Regulation of LET-7 by LIN28B Isoform–Specific Functions

The RNA-binding protein LIN28B plays an important role in development, stem cell biology, and tumorigenesis. LIN28B has two isoforms: the LIN28B-long and -short isoforms. Although studies have revealed the functions of the LIN28B-long isoform in tumorigenesis, the role of the LIN28B-short isoform remains unclear and represents a major gap in the field. The LIN28B-long and -short isoforms are expressed in a subset of human colorectal cancers and adjacent normal colonic mucosa, respectively. To elucidate the functional and mechanistic aspects of these isoforms, colorectal cancer cells (Caco-2 and LoVo) were generated to either express no LIN28B or the -short or -long isoform. Interestingly, the long isoform suppressed LET-7 expression and activated canonical RAS/ERK signaling, whereas the short isoform did not. The LIN28B-long isoform–expressing cells demonstrated increased drug resistance to 5-fluorouracil and cisplatin through the upregulation of ERCC1, a DNA repair gene, in a LET-7–dependent manner. The LIN28B-short isoform preserved its ability to bind pre-let-7, without inhibiting the maturation of LET-7, and competed with the LIN28B-long isoform for binding to pre-let-7. Coexpression of the short isoform in the LIN28B-long isoform–expressing cells rescued the phenotypes induced by the LIN28B-long isoform. Implications: This study demonstrates the differential antagonistic functions of the LIN28B-short isoform against the LIN28B-long isoform through an inability to degrade LET-7, which leads to the novel premise that the short isoform may serve to counterbalance the long isoform during normal colonic epithelial homeostasis, but its downregulation during colonic carcinogenesis may reveal the protumorigenic effects of the long isoform. Mol Cancer Res; 16(3); 403–16. ©2018 AACR.

RNA structure, binding, and coordination in Arabidopsis

From the moment of transcription, up through degradation, each RNA transcript is bound by an ever‐changing cohort of RNA binding proteins. The binding of these proteins is regulated by both the primary RNA sequence, as well as the intramolecular RNA folding, or secondary structure, of the transcript. Thus, RNA secondary structure regulates many post‐transcriptional processes. With the advent of next generation sequencing, several techniques have been developed to generate global landscapes of both RNA–protein interactions and RNA secondary structure. In this review, we describe the current state of the field detailing techniques to globally interrogate RNA secondary structure and/or RNA–protein interaction sites, as well as our current understanding of these features in the transcriptome of the model plant Arabidopsis thaliana. WIREs RNA 2017, 8:e1426. doi: 10.1002/wrna.1426

Protein Interaction Profile Sequencing (PIP‐seq)

Every eukaryotic RNA transcript undergoes extensive post‐transcriptional processing from the moment of transcription up through degradation. This regulation is performed by a distinct cohort of RNA‐binding proteins which recognize their target transcript by both its primary sequence and secondary structure. Here, we describe protein interaction profile sequencing (PIP‐seq), a technique that uses ribonuclease‐based footprinting followed by high‐throughput sequencing to globally assess both protein‐bound RNA sequences and RNA secondary structure. PIP‐seq utilizes single‐ and double‐stranded RNA‐specific nucleases in the absence of proteins to infer RNA secondary structure. These libraries are also compared to samples that undergo nuclease digestion in the presence of proteins in order to find enriched protein‐bound sequences. Combined, these four libraries provide a comprehensive, transcriptome‐wide view of RNA secondary structure and RNA protein interaction sites from a single experimental technique.

Isolation and sequencing of AGO-bound RNAs reveals characteristics of mammalian stem-loop processing in vivo

MicroRNA precursors (pre-miRNAs) are short hairpin RNAs that are rapidly processed into mature microRNAs (miRNAs) in the cytoplasm. Due to their low abundance in cells, sequencing-based studies of pre-miRNAs have been limited. We successfully enriched for and deep sequenced pre-miRNAs in human cells by capturing these RNAs during their interaction with Argonaute (AGO) proteins. Using this approach, we detected > 350 pre-miRNAs in human cells and > 250 pre-miRNAs in a reanalysis of a similar study in mouse cells. We uncovered widespread trimming and non-templated additions to the 3’ ends of pre- and mature miRNAs. Additionally, we created an index for microRNA precursor processing efficiency. This analysis revealed a subset of pre-miRNAs that produce low levels of mature miRNAs despite abundant precursors, including an annotated miRNA in the 5’ UTR of the DiGeorge syndrome critical region 8 mRNA transcript. This led us to search for AGO-associated stem-loops originating from other mRNA species, which identified hundreds of putative pre-miRNAs derived from human and mouse mRNAs. In summary, we provide a wealth of information on mammalian pre-miRNAs, and identify novel microRNA and microRNA-like elements localized in mRNAs.

The LIN28B–IMP1 post-transcriptional regulon has opposing effects on oncogenic signaling in the intestine

RNA-binding proteins (RBPs) are expressed broadly during both development and malignant transformation, yet their mechanistic roles in epithelial homeostasis or as drivers of tumor initiation and progression are incompletely understood. Here we describe a novel interplay between RBPs LIN28B and IMP1 in intestinal epithelial cells. Ribosome profiling and RNA sequencing identified IMP1 as a principle node for gene expression regulation downstream from LIN28B In vitro and in vivo data demonstrate that epithelial IMP1 loss increases expression of WNT target genes and enhances LIN28B-mediated intestinal tumorigenesis, which was reversed when we overexpressed IMP1 independently in vivo. Furthermore, IMP1 loss in wild-type or LIN28B-overexpressing mice enhances the regenerative response to irradiation. Together, our data provide new evidence for the opposing effects of the LIN28B-IMP1 axis on post-transcriptional regulation of canonical WNT signaling, with implications in intestinal homeostasis, regeneration and tumorigenesis.