Biochemical Analysis of Post-Transcriptional Regulatory Mechanisms
Post-transcriptional regulation of mRNA stability and translation by noncoding RNAs (ncRNAs) and RNA-binding proteins (RBPs) represents a vital cellular process. Deregulation of the mRNA-RBP and mRNA-ncRNA interactions by genetic mutation, deletion, or dysregulation results in human diseases. The identification of the RNA target sites of ncRNAs and RBPs relevant to the phenotypic changes observed in disease is a difficult undertaking because of the complexity of the underlying interaction networks. We developed photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) to capture RBP-RNA interactions followed by deep-sequencing of cDNA libraries prepared from their crosslinked RNA segments. Cells are first cultured in 4-thiouridine-(4SU-)–supplemented medium, followed by UV365-nm irradiation of live cells for crosslinking 4SU-modified RNA bound by RBPs. Cross-linked RBP-RNA segments are subsequently isolated by immunoprecipitation from cell lysates, and the cross-linked RNAs are subjected to small RNA cDNA library preparation and deep sequencing. The resulting sequence reads are mapped to the genome and transcriptome, and clusters of sequence reads with T-to-C sequence transitions represent 4SU-crosslinked RNA-binding sites.
We have applied this method to determine the RNA targets and underlying RNA recognition elements for many families of RBPs, including AGO, IGF2BP, QKI, PUM, FUS, ELAVL, FMR1, LIN28, and RBPMS. PAR-CLIP typically yields thousands to tens of thousands of RNA-binding sites in support of complex RNA interaction networks. These natural RNA-binding sites also represent excellent ligands for further biochemical and collaborative structural studies conducted by Dinshaw Patel’s laboratory at Memorial Sloan-Kettering Cancer Center (MSKCC).
To assess the regulatory function of target RNA binding and the physiologic consequences, we monitor global changes in RNA and protein abundance in the same cell systems established for defining the RNA targets upon overexpression or small interfering RNA (siRNA) knockdown and CRISPR knockout of the selected RBP. Subsequent correlation of regulated to PAR-CLIP–identified targets reveals critical RNA sequence and context features and allows for the ranking of RNA targets for follow-up studies. Some of the complex bioinformatic analysis is carried out in collaboration with Uwe Ohler’s laboratory at the Max-Delbrueck Center in Berlin. Specific phenotypic studies of regulatory functions of RBPs may be conducted in animal models.
A census of RBPs by our laboratory established that the human genome encodes close to 1,550, several hundred of which represent mRNA-binding proteins contributing to pre-mRNA processing, transport, stability, and translation, as well as rRNA- and tRNA-binding proteins. One of the long-term aims of my laboratory is to determine a complete RNA-RBP interactome and to develop cell-based and molecular assays to elucidate the specific regulatory and basic molecular functions of mRNA-binding proteins. To this aim, we have initiated a human transcriptome curation project, which will provide non-redundant reference transcripts to all human genes and their allelic variation.
Characterization of Noncoding RNAs
Our interest in post-transcriptional gene regulation was triggered by the discovery of short double-stranded RNAs (dsRNAs) as sequence-specific repressors of gene expression in processes known as RNA silencing and RNA interference. We defined the size and structure of the dsRNA-processing intermediates, the siRNAs, and studied their assembly into target RNA-binding and -cleaving ribonucleoprotein complexes. In this process, we also discovered hundreds of endogenous small RNAs implicated in gene regulation.
The most abundant class of small ncRNAs have been termed microRNAs (miRNAs); these are encoded in the genome in the form of short, slightly imperfect inverted repeats of approximately 25 base pair lengths. Mature miRNAs are predominantly 21 or 22 nucleotides (nt) long, evolutionarily conserved, and present in every cell type. Humans express several hundred miRNA genes acting as negative regulators of target mRNA expression. A small subset of these miRNA genes are expressed with cell-type and developmental stage specificity and contribute to establishment or maintenance of cell lineages.
The second class of small ncRNAs were termed piRNAs; they are 26 to 32 nt long, begin with a 5’ uridine, and are 2’-O-methylated at their 3 ends. In contrast to miRNAs, which originate from dsRNA precursors, piRNAs are processed from long, single-stranded, nonconserved primary transcripts. They are specifically expressed in female embryonic and adult male germ cells during germ cell expansion and are essential in male for fertility and have been implicated in protection against transposable elements. The human genome encodes about 150 piRNA-producing genes, yielding hundreds of thousands of sequence-distinct piRNAs.
Finally, many longer ncRNAs (50-200 nt), such as tRNAs, snRNAs, and snoRNAs, have not been fully characterized in humans. For their capture and curation, we adapted our RNA-sequencing approaches, and were able to detect tRNA splicing defects in patients with disease-causing mutations in a tRNA splicing factor.
Nucleic Acid Sensing Pathways in Innate Immunity
The detection of aberrant nucleic acid, aberrant referring to distinct in localization and chemical modification from mature endogenous cellular nucleic acids, is coordinated by specialized nucleic acid binding proteins either expressed in all cells or within specialized cells of the immune system and triggering a variety of cellular stress and innate immunity responses such as induction of interferon expression. We have co-discovered the second messenger cyclic dinucleotide c[G(2′,5′)pA(3′,5′)p] or cGAMP, which is synthesized from ATP and GTP by the cytosolic dsDNA-dependent nucleotidyltransferase MB21D1/cGAS. cGAMP binds to the ER membrane-anchored receptor STING, which initiates a phosphorylation cascade ultimately leading to transcriptional activation of interferon genes in macrophages and dendritic cells. We have since developed small molecule inhibitors and activators of the pathway in collaboration with Fraser Glickman’s laboratory at the Rockefeller High Throughput Screening Resource Center and structural biologist Dinshaw Patel at MSKCC for treatment of autoimmune diseases or as cancer vaccine, respectively.
Future RNA Diagnostic Developments
We continue to characterize the expression RNA in normal and disease states in tissues and cells, and extended our efforts monitoring their presence in extracellular body fluids such as serum, plasma, and urine to evaluate their utility as biomarkers and in diagnostics. As clinical studies require processing of hundreds to thousands of clinical specimens, we first developed automated DNA- and RNA isolation protocols for recovering protein- and microvesicle-protected RNA from otherwise nuclease-rich cell-free blood and urine samples. In collaboration with clinical laboratories focused on chronic heart, liver and kidney diseases we have processed large sample collections and characterized these exRNAs by deep sequencing and documented the utility of certain miRNAs as biomarker of disease. To take advantage of all RNA fragments present in bodily fluids, we are optimizing methods and bioinformatic analysis towards capturing mRNA fragments, which we consider more specific indicators for their originating cell type as well as disease processes.
Along with our interests in RNA diagnostics, we continue to develop multicolor fluorescence RNA in situ hybridization (RNA FISH) using formalin-fixed, paraffin-embedded archival tissue sections by enhancing RNA fixation, oligonucleotide probe specificity, signal amplification techniques. These studies are paired with single-cell RNA sequencing methodologies (Fluidigm and DropSeq) using human kidney and skin biopsies of Lupus patients, aimed at discovering diagnostic and prognostic signatures and supporting tissue-based pathology reports.
These studies are performed in collaboration with the laboratories of Jill Buyon at the NYU Langone Medical Center, Manikkam Suthanthiran at the Weill Cornell Medical College, Beatrice Goilav at the Children’s Hospital at Montefiore, Chaim Putterman at the Albert Einstein College of Medicine, and Markus Bitzer at the University of Michigan Health System.
Grants from the National Institutes of Health, Starr Foundation, Simons Foundation, and Robertson Therapeutic Development Fund provided partial support for these projects.
As of February 22, 2016