Molly Hammell

Organoid Models of Human and Mouse Ductal Pancreatic Cancer

Pancreatic cancer is one of the most lethal malignancies due to its late diagnosis and limited response to treatment. Tractable methods to identify and interrogate pathways involved in pancreatic tumorigenesis are urgently needed. We established organoid models from normal and neoplastic murine and human pancreas tissues. Pancreatic organoids can be rapidly generated from resected tumors and biopsies, survive cryopreservation, and exhibit ductal- and disease-stage-specific characteristics. Orthotopically transplanted neoplastic organoids recapitulate the full spectrum of tumor development by forming early-grade neoplasms that progress to locally invasive and metastatic carcinomas. Due to their ability to be genetically manipulated, organoids are a platform to probe genetic cooperation. Comprehensive transcriptional and proteomic analyses of murine pancreatic organoids revealed genes and pathways altered during disease progression. The confirmation of many of these protein changes in human tissues demonstrates that organoids are a facile model system to discover characteristics of this deadly malignancy.

mirWIP: microRNA target prediction based on microRNA-containing ribonucleoprotein-enriched transcripts

Target prediction for animal microRNAs (miRNAs) has been hindered by the small number of verified targets available to evaluate the accuracy of predicted miRNA-target interactions. Recently, a dataset of 3,404 miRNA-associated mRNA transcripts was identified by immunoprecipitation of the RNA-induced silencing complex components AIN-1 and AIN-2. Our analysis of this AIN-IP dataset revealed enrichment for defining characteristics of functional miRNA-target interactions, including structural accessibility of target sequences, total free energy of miRNA-target hybridization and topology of base-pairing to the 5′ seed region of the miRNA. We used these enriched characteristics as the basis for a quantitative miRNA target prediction method, miRNA targets by weighting immunoprecipitation-enriched parameters (mirWIP), which optimizes sensitivity to verified miRNA-target interactions and specificity to the AIN-IP dataset. MirWIP can be used to capture all known conserved miRNA-mRNA target relationships in Caenorhabditis elegans at a lower false-positive rate than can the current standard methods.

Multiple roles for Piwi in silencing Drosophila transposons

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.

piRNA-directed cleavage of meiotic transcripts regulates spermatogenesis

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.

Transposable Elements in TDP-43-Mediated Neurodegenerative Disorders

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.

Computational methods to identify miRNA targets

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.

Effect of life history on microRNA expression during C. elegans development

Animals have evolved mechanisms to ensure the robustness of developmental outcomes to changing environments. MicroRNA expression may contribute to developmental robustness because microRNAs are key post-transcriptional regulators of developmental gene expression and can affect the expression of multiple target genes. Caenorhabditis elegans provides an excellent model to study developmental responses to environmental conditions. In favorable environments, C. elegans larvae develop rapidly and continuously through four larval stages. In contrast, in unfavorable conditions, larval development may be interrupted at either of two diapause stages: The L1 diapause occurs when embryos hatch in the absence of food, and the dauer diapause occurs after the second larval stage in response to environmental stimuli encountered during the first two larval stages. Dauer larvae are stress resistant and long lived, permitting survival in harsh conditions. When environmental conditions improve, dauer larvae re-enter development, and progress through two post-dauer larval stages to adulthood. Strikingly, all of these life history options (whether continuous or interrupted) involve an identical pattern and sequence of cell division and cell fates. To identify microRNAs with potential functions in buffering development in the context of C. elegans life history options, we used multiplex real-time PCR to assess the expression of 107 microRNAs throughout development in both continuous and interrupted life histories. We identified 17 microRNAs whose developmental profile of expression is affected by dauer life history and/or L1 diapause, compared to continuous development. Hence these microRNAs could function to regulate gene expression programs appropriate for different life history options in the developing worm.

Systematic analysis of dynamic miRNA-target interactions during C. elegans development.

Although microRNA (miRNA)-mediated functions have been implicated in many aspects of animal development, the majority of miRNA::mRNA regulatory interactions remain to be characterized experimentally. We used an AIN/GW182 protein immunoprecipitation approach to systematically analyze miRNA::mRNA interactions during C. elegans development. We characterized the composition of miRNAs in functional miRNA-induced silencing complexes (miRISCs) at each developmental stage and identified three sets of miRNAs with distinct stage-specificity of function. We then identified thousands of miRNA targets in each developmental stage, including a significant portion that is subject to differential miRNA regulation during development. By identifying thousands of miRNA family-mRNA pairs with temporally correlated patterns of AIN-2 association, we gained valuable information on the principles of physiological miRNA::target recognition and predicted 1589 high-confidence miRNA family::mRNA interactions. Our data support the idea that miRNAs preferentially target genes involved in signaling processes and avoid genes with housekeeping functions, and that miRNAs orchestrate temporal developmental programs by coordinately targeting or avoiding genes involved in particular biological functions.

The Chemical Distribution in a Subluminous Type Ia Supernova: Hubble Space Telescope Images of the SN 1885 Remnant*

SN 1885 was a probable subluminous Type Ia supernova which occurred in the bulge of the Andromeda galaxy, M31, at a projected location 16 from the nucleus. Here we present and analyze Hubble Space Telescope images of the SN 1885 remnant seen in absorption against the M31 bulge via the resonance lines of Ca I, Ca II, Fe I, and Fe II. Viewed in Ca II H & K line absorption, the remnant appears as a nearly black circular spot with an outermost angular radius of 0. 40 ± 0. 025, implying a maximum linear radius of 1.52± 0.15 pc at M31’s estimated distance of 785± 30 kpc and hence a 120 yr average expansion velocity of 12,400± 1400 kms. The strongest Ca II absorption is organized in a broken ring structure with a radius of 0. 2 (= 6000 km s) with several apparent absorption ‘clumps’ of an angular size around that of the image pixel scale of 0. 05 (= 1500 km s). Ca I and Fe I absorption structures appear similar except for a small Fe I absorption peak displaced 0. 1 off-center of the Ca II structure by a projected velocity of about 3000 km s. Analyses of these images using off-center, delayed-detonation models suggest a low Ni production similar to the subluminous SN Ia explosion of SN 1986G. The strongly lopsided images of of Ca I and Fe I can be understood as resulting from an aspherical chemical distribution, with the best agreement found using an off-center model viewed from an inclination of ∼ 60. The detection of small scale Ca II clumps is the first direct evidence for some instabilities and the existence of a deflagration phase in SNe Ia or, alternatively, mixing induced by radioactive decay of Ni over time scales of seconds or days. However, the degree of mixing allowed by the observed images is much smaller than current 3D calculations for Rayleigh-Taylor dominated deflagration fronts. Moreover, the images require a central region of no or little Ca but iron group elements indicative for burning under sufficiently high densities for electron capture taking place, i.e., burning prior to a significant pre-expansion of the WD. Using time-dependent ionization calculations, we show that the presence today of neutral ions in this 120 yr old remnant can be understood as ejecta self-shielding from the UV radiation in the M31 bulge. Subject headings: supernovae: general supernovae: individual (SN 1885) ISM: kinematics and dynamics ISM: abundances supernova remnants

An atlas of chromatoid body components

The genome of male germ cells is actively transcribed during spermatogenesis to produce phase-specific protein-coding mRNAs and a considerable amount of different noncoding RNAs. Ribonucleoprotein (RNP) granule-mediated RNA regulation provides a powerful means to secure the quality and correct expression of the requisite transcripts. Haploid spermatids are characterized by a unique, unusually large cytoplasmic granule, the chromatoid body (CB), which emerges during the switch between the meiotic and post-meiotic phases of spermatogenesis. To better understand the role of the CB in male germ cell differentiation, we isolated CBs from mouse testes and revealed its full RNA and protein composition. We showed that the CB is mainly composed of RNA-binding proteins and other proteins involved RNA regulation. The CB was loaded with RNA, including pachytene piRNAs, a diverse set of mRNAs, and a number of uncharacterized long noncoding transcripts. The CB was demonstrated to accumulate nascent RNA during all the steps of round spermatid differentiation. Our results revealed the CB as a large germ cell-specific RNP platform that is involved in the control of the highly complex transcriptome of haploid male germ cells.