MicroRNAs (miRNAs) are short RNA molecules that regulate the post-transcriptional expression of their target genes. This regulation may take the form of stable translational or degradation of the ...target transcript, although the mechanisms governing the outcome of miRNA-mediated regulation remain largely unknown. While it is becoming clear that miRNAs are core components of gene regulatory networks, elucidating precise roles for each miRNA within these networks will require an accurate means of identifying target genes and assessing the impact of miRNAs on individual targets. Numerous computational methods for predicting targets are currently available. These methods vary widely in their emphasis, accuracy, and ease of use for researchers. This review will focus on a comparison of the available computational methods in animals, with an emphasis on approaches that are informed by experimental analysis of microRNA:target complexes.
Most RNA-seq data analysis software packages are not designed to handle the complexities involved in properly apportioning short sequencing reads to highly repetitive regions of the genome. These ...regions are often occupied by transposable elements (TEs), which make up between 20 and 80% of eukaryotic genomes. They can contribute a substantial portion of transcriptomic and genomic sequence reads, but are typically ignored in most analyses.
Here, we present a method and software package for including both gene- and TE-associated ambiguously mapped reads in differential expression analysis. Our method shows improved recovery of TE transcripts over other published expression analysis methods, in both synthetic data and qPCR/NanoString-validated published datasets.
The source code, associated GTF files for TE annotation, and testing data are freely available at http://hammelllab.labsites.cshl.edu/software.
mhammell@cshl.edu.
Supplementary data are available at Bioinformatics online.
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.
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.
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive loss of motor neurons. While several pathogenic mutations have been identified, the vast ...majority of ALS cases have no family history of disease. Thus, for most ALS cases, the disease may be a product of multiple pathways contributing to varying degrees in each patient. Using machine learning algorithms, we stratify the transcriptomes of 148 ALS postmortem cortex samples into three distinct molecular subtypes. The largest cluster, identified in 61% of patient samples, displays hallmarks of oxidative and proteotoxic stress. Another 19% of the samples shows predominant signatures of glial activation. Finally, a third group (20%) exhibits high levels of retrotransposon expression and signatures of TARDBP/TDP-43 dysfunction. We further demonstrate that TDP-43 (1) directly binds a subset of retrotransposon transcripts and contributes to their silencing in vitro, and (2) pathological TDP-43 aggregation correlates with retrotransposon de-silencing in vivo.
Display omitted
•ALS patient cortex samples can be grouped into 3 distinct expression profiles•Aberrant pathways include stress, inflammation, and TDP-43 pathology•Elevated retrotransposon expression is correlated with TDP-43 pathology•TDP-43 directly binds retrotransposons in cells and contributes to their silencing
Tam et al. present transcriptome profiling results from a large set of amyotrophic lateral sclerosis (ALS) patient cortex samples, finding 3 distinct groups. Two ALS subtypes are marked by gene pathways previously associated with ALS disease, while a third group shows elevated retrotransposon expression linked to TDP-43 pathology.
Silencing of transposons in the Drosophila ovary relies on three Piwi family proteins--Piwi, Aubergine (Aub), and Ago3--acting in concert with their small RNA guides, the Piwi-interacting RNAs ...(piRNAs). Aub and Ago3 are found in the germ cell cytoplasm, where they function in the ping-pong cycle to consume transposon mRNAs. The nuclear Piwi protein is required for transposon silencing in both germ and somatic follicle cells, yet the precise mechanisms by which Piwi acts remain largely unclear. We investigated the role of Piwi by combining cell type-specific knockdowns with measurements of steady-state transposon mRNA levels, nascent RNA synthesis, chromatin state, and small RNA abundance. In somatic cells, Piwi loss led to concerted effects on nascent transcripts and transposon mRNAs, indicating that Piwi acts through transcriptional gene silencing (TGS). In germ cells, Piwi loss showed disproportionate impacts on steady-state RNA levels, indicating that it also exerts an effect on post-transcriptional gene silencing (PTGS). Piwi knockdown affected levels of germ cell piRNAs presumably bound to Aub and Ago3, perhaps explaining its post-transcriptional impacts. Overall, our results indicate that Piwi plays multiple roles in the piRNA pathway, in part enforcing transposon repression through effects on local chromatin states and transcription but also participating in germ cell piRNA biogenesis.
Elevated expression of specific transposable elements (TEs) has been observed in several neurodegenerative disorders. TEs also can be active during normal neurogenesis. By mining a series of deep ...sequencing datasets of protein-RNA interactions and of gene expression profiles, we uncovered extensive binding of TE transcripts to TDP-43, an RNA-binding protein central to amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Second, we find that association between TDP-43 and many of its TE targets is reduced in FTLD patients. Third, we discovered that a large fraction of the TEs to which TDP-43 binds become de-repressed in mouse TDP-43 disease models. We propose the hypothesis that TE mis-regulation contributes to TDP-43 related neurodegenerative diseases.
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.
MIWI catalytic activity is required for spermatogenesis, indicating that piRNA-guided cleavage is critical for germ cell development. To identify meiotic piRNA targets, we augmented the mouse piRNA ...repertoire by introducing a human meiotic piRNA cluster. This triggered a spermatogenesis defect by inappropriately targeting the piRNA machinery to mouse mRNAs essential for germ cell development. Analysis of such de novo targets revealed a signature for pachytene piRNA target recognition. This enabled identification of both transposable elements and meiotically expressed protein-coding genes as targets of native piRNAs. Cleavage of genic targets began at the pachytene stage and resulted in progressive repression through meiosis, driven at least in part via the ping-pong cycle. Our data support the idea that meiotic piRNA populations must be strongly selected to enable successful spermatogenesis, both driving the response away from essential genes and directing the pathway toward mRNA targets that are regulated by small RNAs in meiotic cells.
Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) are two incurable neurodegenerative disorders that exist on a symptomological spectrum and share both genetic ...underpinnings and pathophysiological hallmarks. Functional abnormality of TAR DNA-binding protein 43 (TDP-43), an aggregation-prone RNA and DNA binding protein, is observed in the vast majority of both familial and sporadic ALS cases and in ~40% of FTLD cases, but the cascade of events leading to cell death are not understood. We have expressed human TDP-43 (hTDP-43) in Drosophila neurons and glia, a model that recapitulates many of the characteristics of TDP-43-linked human disease including protein aggregation pathology, locomotor impairment, and premature death. We report that such expression of hTDP-43 impairs small interfering RNA (siRNA) silencing, which is the major post-transcriptional mechanism of retrotransposable element (RTE) control in somatic tissue. This is accompanied by de-repression of a panel of both LINE and LTR families of RTEs, with somewhat different elements being active in response to hTDP-43 expression in glia versus neurons. hTDP-43 expression in glia causes an early and severe loss of control of a specific RTE, the endogenous retrovirus (ERV) gypsy. We demonstrate that gypsy causes the degenerative phenotypes in these flies because we are able to rescue the toxicity of glial hTDP-43 either by genetically blocking expression of this RTE or by pharmacologically inhibiting RTE reverse transcriptase activity. Moreover, we provide evidence that activation of DNA damage-mediated programmed cell death underlies both neuronal and glial hTDP-43 toxicity, consistent with RTE-mediated effects in both cell types. Our findings suggest a novel mechanism in which RTE activity contributes to neurodegeneration in TDP-43-mediated diseases such as ALS and FTLD.
PDF
