Faculty Bibliography
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49
Decoding the 5' nucleotide bias of PIWI-interacting RNAs
PIWI-interacting RNAs (piRNAs) are at the center of a small RNA-based immune system that defends genomes against the deleterious action of mobile genetic elements (transposons). PiRNAs are highly variable in sequence with extensive targeting potential. Their diversity is restricted by their preference to start with a Uridine (U) at the 5' most position (1U-bias), a bias that remains poorly understood. Here we uncover that the 1U-bias of Piwi-piRNAs is established by consecutive discrimination against all nucleotides but U, first during piRNA biogenesis and then upon interaction with Piwi's specificity loop. Sequence preferences during piRNA processing also restrict U across the piRNA body with the potential to directly impact target recognition. Overall, the uncovered signatures could modulate specificity and efficacy of piRNA-mediated transposon restriction, and provide a substrate for purifying selection in the ongoing arms race between genomes and their mobile parasites.
PMCID:6381166
PMID: 30783109
ISSN: 2041-1723
CID: 5428502
Diseases of the nERVous system: retrotransposon activity in neurodegenerative disease
Transposable Elements (TEs) are mobile genetic elements whose sequences constitute nearly half of the human genome. Each TE copy can be present in hundreds to thousands of locations within the genome, complicating the genetic and genomic studies of these highly repetitive sequences. The recent development of better tools for evaluating TE derived sequences in genomic studies has enabled an increasing appreciation for the contribution of TEs to human development and disease. While some TEs have contributed novel and beneficial host functions, this review will summarize the evidence for detrimental TE activity in neurodegenerative disorders. Much of the evidence for pathogenicity implicates endogenous retroviruses (ERVs), a subset of TEs that entered the genome by retroviral infections of germline cells in our evolutionary ancestors and have since been passed down as a substantial fraction of the human genome. Human specific ERVs (HERVs) represent some of the youngest ERVs in the genome, and thus are presumed to retain greater function and resultant pathogenic potential.
PMCID:6659213
PMID: 31372185
ISSN: 1759-8753
CID: 5428182
Ten things you should know about transposable elements
Transposable elements (TEs) are major components of eukaryotic genomes. However, the extent of their impact on genome evolution, function, and disease remain a matter of intense interrogation. The rise of genomics and large-scale functional assays has shed new light on the multi-faceted activities of TEs and implies that they should no longer be marginalized. Here, we introduce the fundamental properties of TEs and their complex interactions with their cellular environment, which are crucial to understanding their impact and manifold consequences for organismal biology. While we draw examples primarily from mammalian systems, the core concepts outlined here are relevant to a broad range of organisms.
PMCID:6240941
PMID: 30454069
ISSN: 1474-760x
CID: 5428482
Single-cell RNA-seq analysis identifies markers of resistance to targeted BRAF inhibitors in melanoma cell populations
Single-cell RNA-seq's (scRNA-seq) unprecedented cellular resolution at a genome-wide scale enables us to address questions about cellular heterogeneity that are inaccessible using methods that average over bulk tissue extracts. However, scRNA-seq data sets also present additional challenges such as high transcript dropout rates, stochastic transcription events, and complex population substructures. Here, we present a single-cell RNA-seq analysis and klustering evaluation (SAKE), a robust method for scRNA-seq analysis that provides quantitative statistical metrics at each step of the analysis pipeline. Comparing SAKE to multiple single-cell analysis methods shows that most methods perform similarly across a wide range of cellular contexts, with SAKE outperforming these methods in the case of large complex populations. We next applied the SAKE algorithms to identify drug-resistant cellular populations as human melanoma cells respond to targeted BRAF inhibitors (BRAFi). Single-cell RNA-seq data from both the Fluidigm C1 and 10x Genomics platforms were analyzed with SAKE to dissect this problem at multiple scales. Data from both platforms indicate that BRAF inhibitor-resistant cells can emerge from rare populations already present before drug application, with SAKE identifying both novel and known markers of resistance. These experimentally validated markers of BRAFi resistance share overlap with previous analyses in different melanoma cell lines, demonstrating the generality of these findings and highlighting the utility of single-cell analysis to elucidate mechanisms of BRAFi resistance.
PMCID:6120620
PMID: 30061114
ISSN: 1549-5469
CID: 5428462
Chromatin-mediated translational control is essential for neural cell fate specification
Neural cell fate specification is a multistep process in which stem cells undergo sequential changes in states, giving rise to particular lineages such as neurons and astrocytes. This process is accompanied by dynamic changes of chromatin and in transcription, thereby orchestrating lineage-specific gene expression programs. A pressing question is how these events are interconnected to sculpt cell fate. We show that altered chromatin due to loss of the chromatin remodeler Chd5 causes neural stem cell activation to occur ahead of time. This premature activation is accompanied by transcriptional derepression of ribosomal subunits, enhanced ribosome biogenesis, and increased translation. These untimely events deregulate cell fate decisions, culminating in the generation of excessive numbers of astrocytes at the expense of neurons. By monitoring the proneural factor Mash1, we further show that translational control is crucial for appropriate execution of cell fate specification, thereby providing new insight into the interplay between transcription and translation at the initial stages of neurogenesis.
PMCID:6238594
PMID: 30456361
ISSN: 2575-1077
CID: 5428492
Unexpected similarities between C9ORF72 and sporadic forms of ALS/FTD suggest a common disease mechanism
Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) represent two ends of a disease spectrum with shared clinical, genetic and pathological features. These include near ubiquitous pathological inclusions of the RNA-binding protein (RBP) TDP-43, and often the presence of a GGGGCC expansion in the C9ORF72 (C9) gene. Previously, we reported that the sequestration of hnRNP H altered the splicing of target transcripts in C9ALS patients (Conlon et al., 2016). Here, we show that this signature also occurs in half of 50 postmortem sporadic, non-C9 ALS/FTD brains. Furthermore, and equally surprisingly, these 'like-C9' brains also contained correspondingly high amounts of insoluble TDP-43, as well as several other disease-related RBPs, and this correlates with widespread global splicing defects. Finally, we show that the like-C9 sporadic patients, like actual C9ALS patients, were much more likely to have developed FTD. We propose that these unexpected links between C9 and sporadic ALS/FTD define a common mechanism in this disease spectrum.
PMID: 30003873
ISSN: 2050-084x
CID: 5429232
Genome-wide Analyses Identify KIF5A as a Novel ALS Gene
To identify novel genes associated with ALS, we undertook two lines of investigation. We carried out a genome-wide association study comparing 20,806 ALS cases and 59,804 controls. Independently, we performed a rare variant burden analysis comparing 1,138 index familial ALS cases and 19,494 controls. Through both approaches, we identified kinesin family member 5A (KIF5A) as a novel gene associated with ALS. Interestingly, mutations predominantly in the N-terminal motor domain of KIF5A are causative for two neurodegenerative diseases: hereditary spastic paraplegia (SPG10) and Charcot-Marie-Tooth type 2 (CMT2). In contrast, ALS-associated mutations are primarily located at the C-terminal cargo-binding tail domain and patients harboring loss-of-function mutations displayed an extended survival relative to typical ALS cases. Taken together, these results broaden the phenotype spectrum resulting from mutations in KIF5A and strengthen the role of cytoskeletal defects in the pathogenesis of ALS.
PMCID:5867896
PMID: 29566793
ISSN: 1097-4199
CID: 5429242
TEsmall Identifies Small RNAs Associated With Targeted Inhibitor Resistance in Melanoma
MicroRNAs (miRNAs) are small 21-22 nt RNAs that act to regulate the expression of mRNA target genes through direct binding to mRNA targets. While miRNAs typically dominate small RNA (sRNA) transcriptomes, many other classes are present including tRNAs, snoRNAs, snRNAs, Y-RNAs, piRNAs, and siRNAs. Interactions between processing machinery and targeting networks of these various sRNA classes remains unclear, largely because these sRNAs are typically analyzed separately. Here, we present TEsmall, a tool that allows for the simultaneous processing and analysis of sRNAs from each annotated class in a single integrated workflow. The pipeline begins with raw fastq reads and proceeds all the way to producing count tables formatted for differential expression analysis. Several interactive charts are also produced to look at overall distributions in length and annotation classes. We next applied the TEsmall pipeline to sRNA libraries generated from melanoma cells responding to targeted inhibitors of the MAPK pathway. Targeted oncogene inhibitors have emerged as way to tailor cancer therapies to the particular mutations present in a given tumor. While these targeted strategies are typically effective for short intervals, the emergence of resistance is extremely common, limiting the effectiveness of single-agent therapeutics and driving the need for a better understanding of resistance mechanisms. Using TEsmall, we identified several microRNAs and other sRNA classes that are enriched in inhibitor resistant melanoma cells in multiple melanoma cell lines and may be able to serve as markers of resistant populations more generally.
PMCID:6186986
PMID: 30349559
ISSN: 1664-8021
CID: 5428472
Analysis of RNA-Seq Data Using TEtranscripts
Transposable elements (TE) are mobile genetic elements that can readily change their genomic position. When not properly silenced, TEs can contribute a substantial portion to the cell's transcriptome, but are typically ignored in most RNA-seq data analyses. One reason for leaving TE-derived reads out of RNA-seq analyses is the complexities involved in properly aligning short sequencing reads to these highly repetitive regions. Here we describe a method for including TE-derived reads in RNA-seq differential expression analysis using an open source software package called TEtranscripts. TEtranscripts is designed to assign both uniquely and ambiguously mapped reads to all possible gene and TE-derived transcripts in order to statistically infer the correct gene/TE abundances. Here, we provide a detailed tutorial of TEtranscripts using a published qPCR validated dataset.Barbara McClintock laid the foundation for TE research with her discoveries in maize of mobile genetic elements capable of inserting into novel locations in the genome, altering the expression of nearby genes [1]. Since then, our appreciation of the contribution of repetitive TE-derived sequences to eukaryotic genomes has vastly increased. With the publication of the first human genome draft by the Human Genome Project, it was determined that nearly half of the human genome is derived from TE sequences [2, 3], with varying levels of repetitive DNA present in most plant and animal species. More recent studies looking at distantly related TE-like sequences have estimated that up to two thirds of the human genome might be repeat-derived [4], with the vast majority of these sequences attributed to retrotransposons that require transcription as part of the mobilization process, as discussed below.
PMID: 29508296
ISSN: 1940-6029
CID: 5428452
Suppression of protein tyrosine phosphatase N23 predisposes to breast tumorigenesis via activation of FYN kinase
Disruption of the balanced modulation of reversible tyrosine phosphorylation has been implicated in the etiology of various human cancers, including breast cancer. Protein Tyrosine Phosphatase N23 (PTPN23) resides in chromosomal region 3p21.3, which is hemizygously or homozygously lost in some breast cancer patients. In a loss-of-function PTPome screen, our laboratory identified PTPN23 as a suppressor of cell motility and invasion in mammary epithelial and breast cancer cells. Now, our TCGA (The Cancer Genome Atlas) database analyses illustrate a correlation between low PTPN23 expression and poor survival in breast cancers of various subtypes. Therefore, we investigated the tumor-suppressive function of PTPN23 in an orthotopic transplantation mouse model. Suppression of PTPN23 in Comma 1Dβ cells induced breast tumors within 56 wk. In PTPN23-depleted tumors, we detected hyperphosphorylation of the autophosphorylation site tyrosine in the SRC family kinase (SFK) FYN as well as Tyr142 in β-catenin. We validated the underlying mechanism of PTPN23 function in breast tumorigenesis as that of a key phosphatase that normally suppresses the activity of FYN in two different models. We demonstrated that tumor outgrowth from PTPN23-deficient BT474 cells was suppressed in a xenograft model in vivo upon treatment with AZD0530, an SFK inhibitor. Furthermore, double knockout of FYN and PTPN23 via CRISPR/CAS9 also attenuated tumor outgrowth from PTPN23 knockout Cal51 cells. Overall, this mechanistic analysis of the tumor-suppressive function of PTPN23 in breast cancer supports the identification of FYN as a therapeutic target for breast tumors with heterozygous or homozygous loss of PTPN23.
PMCID:5710140
PMID: 29066500
ISSN: 1549-5477
CID: 5428442
