Jongkyu Kim

The ribonuclease activity of SAMHD1 is required for HIV-1 restriction

The HIV-1 restriction factor SAM domain– and HD domain–containing protein 1 (SAMHD1) is proposed to inhibit HIV-1 replication by depleting the intracellular dNTP pool. However, phosphorylation of SAMHD1 regulates its ability to restrict HIV-1 without decreasing cellular dNTP levels, which is not consistent with a role for SAMHD1 dNTPase activity in HIV-1 restriction. Here, we show that SAMHD1 possesses RNase activity and that the RNase but not the dNTPase function is essential for HIV-1 restriction. By enzymatically characterizing Aicardi-Goutières syndrome (AGS)-associated SAMHD1 mutations and mutations in the allosteric dGTP-binding site of SAMHD1 for defects in RNase or dNTPase activity, we identify SAMHD1 point mutants that cause loss of one or both functions. The RNase-positive and dNTPase-negative SAMHD1D137N mutant is able to restrict HIV-1 infection, whereas the RNase-negative and dNTPase-positive SAMHD1Q548A mutant is defective for HIV-1 restriction. SAMHD1 associates with HIV-1 RNA and degrades it during the early phases of cell infection. SAMHD1 silencing in macrophages and CD4+ T cells from healthy donors increases HIV-1 RNA stability, rendering the cells permissive for HIV-1 infection. Furthermore, phosphorylation of SAMHD1 at T592 negatively regulates its RNase activity in cells and impedes HIV-1 restriction. Our results reveal that the RNase activity of SAMHD1 is responsible for preventing HIV-1 infection by directly degrading the HIV-1 RNA.

Genomic portrait of resectable hepatocellular carcinomas: implications of RB1 and FGF19 aberrations for patient stratification.

Hepatic resection is the most curative treatment option for early‐stage hepatocellular carcinoma, but is associated with a high recurrence rate, which exceeds 50% at 5 years after surgery. Understanding the genetic basis of hepatocellular carcinoma at surgically curable stages may enable the identification of new molecular biomarkers that accurately identify patients in need of additional early therapeutic interventions. Whole exome sequencing and copy number analysis was performed on 231 hepatocellular carcinomas (72% with hepatitis B viral infection) that were classified as early‐stage hepatocellular carcinomas, candidates for surgical resection. Recurrent mutations were validated by Sanger sequencing. Unsupervised genomic analyses identified an association between specific genetic aberrations and postoperative clinical outcomes. Recurrent somatic mutations were identified in nine genes, including TP53, CTNNB1, AXIN1, RPS6KA3, and RB1. Recurrent homozygous deletions in FAM123A, RB1, and CDKN2A, and high‐copy amplifications in MYC, RSPO2, CCND1, and FGF19 were detected. Pathway analyses of these genes revealed aberrations in the p53, Wnt, PIK3/Ras, cell cycle, and chromatin remodeling pathways. RB1 mutations were significantly associated with cancer‐specific and recurrence‐free survival after resection (multivariate P = 0.038 and P = 0.012, respectively). FGF19 amplifications, known to activate Wnt signaling, were mutually exclusive with CTNNB1 and AXIN1 mutations, and significantly associated with cirrhosis (P = 0.017). Conclusion: RB1 mutations can be used as a prognostic molecular biomarker for resectable hepatocellular carcinoma. Further study is required to investigate the potential role of FGF19 amplification in driving hepatocarcinogenesis in patients with liver cirrhosis and to investigate the potential of anti‐FGF19 treatment in these patients. (Hepatology 2014;60:1971–1981)

TALEN-based knockout library for human microRNAs

Various technical tools have been developed to probe the functions of microRNAs (miRNAs), yet their application has been limited by low efficacy and specificity. To overcome the limitations, we used transcription activator–like effector nucleases (TALENs) to knock out human miRNA genes. We designed and produced a library of 540 pairs of TALENs for 274 miRNA loci, focusing on potentially important miRNAs. The knockout procedure takes only 2–4 weeks and can be applied to any cell type. As a case study, we generated knockout cells for two related miRNAs, miR-141 and miR-200c, which belong to the highly conserved miR-200 family. Interestingly, miR-141 and miR-200c, despite their overall similarity, suppress largely nonoverlapping groups of targets, thus suggesting that functional miRNA-target interaction requires strict seed-pairing. Our study illustrates the potency of TALEN technology and provides useful resources for miRNA research.

Effect of hippocampus extirpation in the rat on glutamate levels in target structures of hippocampal efferents

Twenty days after complete uni- or bilateral hippocampus extirpation in rats, a 25% decrease in glutamate concentration was observed in the septum. Glutamate content also decreased in other terminal structures of the hippocampo-subicular system, i.e. entorhinal cortex, nucleus accumbens septi, mammillary bodies and contralateral hippocampus. It is concluded that the fall in glutamate content which is absent in caudate nucleus is specific for target regions of the hippocampal efferents, adding further support to the suggested transmitter role of glutamate in the limbic system.

General rules for functional microRNA targeting

The functional rules for microRNA (miRNA) targeting remain controversial despite their biological importance because only a small fraction of distinct interactions, called site types, have been examined among an astronomical number of site types that can occur between miRNAs and their target mRNAs. To systematically discover functional site types and to evaluate the contradicting rules reported previously, we used large-scale transcriptome data and statistically examined whether each of approximately 2 billion site types is enriched in differentially downregulated mRNAs responding to overexpressed miRNAs. Accordingly, we identified seven non-canonical functional site types, most of which are novel, in addition to four canonical site types, while also removing numerous false positives reported by previous studies. Extensive experimental validation and significantly elevated 3′ UTR sequence conservation indicate that these non-canonical site types may have biologically relevant roles. Our expanded catalog of functional site types suggests that the gene regulatory network controlled by miRNAs may be far more complex than currently understood.

The commissural fibers in rabbit hippocampus: synapses and their transmitter.

Publisher Summary This chapter presents an experiment for a complete unilateral hippocampus extirpation in rabbits. It investigates the degree of commissural deafferentation in the contralateral hippocampus by electron microscopy. Glutamate (Glu) levels were determined in single layers of the hippocampal fields, regio superior, regio inferior, and area dentata, 20 days after the operation and compared to those found in sham-operated controls. After commissural deafferentation, Glu content decreased significantly in stratum oriens, but no decreases were observed in the other layers receiving commissural endings. Additionally, Glu level increased in layers containing the supposed Glu-ergic terminals of the entorhinal perforant path fibers and the Schaffers collaterals. To explain these findings, the chapter proposes that Glu is located in the commissural nerve endings, but that as a result of the sprouting of neighboring Glu-ergic terminals of other origin, Glu content may normalize in some of the denervated zones.

Genomic Alterations in the RB Pathway Indicate Prognostic Outcomes of Early-Stage Lung Adenocarcinoma

Purpose: To better understand the complete genomic architecture of lung adenocarcinoma. Experimental Design: We used array experiments to determine copy number variations and sequenced the complete exomes of the 247 lung adenocarcinoma tumor samples along with matched normal cells obtained from the same patients. Fully annotated clinical data were also available, providing an unprecedented opportunity to assess the impact of genomic alterations on clinical outcomes. Results: We discovered that genomic alternations in the RB pathway are associated with significantly shorter disease-free survival in early-stage lung adenocarcinoma patients. This association was also observed in our independent validation cohort. The current treatment guidelines for early-stage lung adenocarcinoma patients recommend follow-up without adjuvant therapy after complete resection, except for high-risk patients. However, our findings raise the interesting possibility that additional clinical interventions might provide medical benefits to early-stage lung adenocarcinoma patients with genomic alterations in the RB pathway. When examining the association between genomic mutation and histologic subtype, we uncovered the characteristic genomic signatures of various histologic subtypes. Notably, the solid and the micropapillary subtypes demonstrated great diversity in the mutated genes, while the mucinous subtype exhibited the most unique landscape. This suggests that a more tailored therapeutic approach should be used to treat patients with lung adenocarcinoma. Conclusions: Our analysis of the genomic and clinical data for 247 lung adenocarcinomas should help provide a more comprehensive genomic portrait of lung adenocarcinoma, define molecular signatures of lung adenocarcinoma subtypes, and lead to the discovery of useful prognostic markers that could be used in personalized treatments for early-stage lung adenocarcinoma patients. Clin Cancer Res; 21(11); 2613–23. ©2014 AACR. See related commentary by Collisson, p. 2418

The SeqAn C++ template library for efficient sequence analysis: A resource for programmers

BACKGROUND The use of novel algorithmic techniques is pivotal to many important problems in life science. For example the sequencing of the human genome (Venter et al., 2001) would not have been possible without advanced assembly algorithms and the development of practical BWT based read mappers have been instrumental for NGS analysis. However, owing to the high speed of technological progress and the urgent need for bioinformatics tools, there was a widening gap between state-of-the-art algorithmic techniques and the actual algorithmic components of tools that are in widespread use. We previously addressed this by introducing the SeqAn library of efficient data types and algorithms in 2008 (Döring et al., 2008). RESULTS The SeqAn library has matured considerably since its first publication 9 years ago. In this article we review its status as an established resource for programmers in the field of sequence analysis and its contributions to many analysis tools. CONCLUSIONS We anticipate that SeqAn will continue to be a valuable resource, especially since it started to actively support various hardware acceleration techniques in a systematic manner.

Global and local competition between exogenously introduced microRNAs and endogenously expressed microRNAs.

It has been reported that exogenously introduced micro-RNA (exo-miRNA) competes with endogenously expressed miRNAs (endo-miRNAs) in human cells, resulting in a detectable upregulation of mRNAs with endo-miRNA target sites (TSs). However, the detailed mechanisms of the competition between exo- and endo-miRNAs remain uninvestigated. In this study, using 74 microarrays that monitored the whole-transcriptome response after introducing miRNAs or siRNAs into HeLa cells, we systematically examined the derepression of mRNAs with exo- and/or endo-miRNA TSs. We quantitatively assessed the effect of the number of endo-miRNA TSs on the degree of mRNA derepression. As a result, we observed that the number of endo-miRNA TSs was significantly associated with the degree of derepression, supporting that the derepression resulted from the competition between exo- and endo-miRNAs. However, when we examined whether the site proficiency of exo-miRNA TSs could also influence mRNA derepression, to our surprise, we discovered a strong positive correlation. Our analysis indicates that site proficiencies of both exo- and endo-miRNA TSs are important determinants for the degree of mRNA derepression, implying that the derepression of mRNAs in response to exo-miRNA is more complex than that currently perceived. Our observations may lead to a more complete understanding of the detailed mechanisms of the competition between exo- and endo-miRNAs and to a more accurate prediction of miRNA targets. Our analysis also suggests an interesting hypothesis that long 3′-UTRs may function as molecular buffer against gene expression regulation by individual miRNAs.

Vaquita: Fast and Accurate Identification of Structural Variation Using Combined Evidence.

Motivation: Comprehensive identification of structural variations (SVs) is a crucial task for studying genetic diversity and diseases. However, it remains challenging. There is only a marginal consensus between different methods, and our understanding of SVs is substantially limited.In general, integration of multiple pieces of evidence including split-read, read-pair, soft-clip, and read-depth yields the best result regarding accuracy. However, doing this step by step is usually cumbersome and computationally expensive. Result: We present Vaquita, an accurate and fast tool for the identification of structural variations, which leverages all four types of evidence in a single program. After merging SVs from split-reads and discordant read-pairs, Vaquita realigns the soft-clipped reads to the selected regions using a fast bit-vector algorithm. Furthermore, it also considers the discrepancy of depth distribution around breakpoints using Kullback-Leibler divergence. Finally, Vaquita provides an additional metric for candidate selection based on voting, and also provides robust prioritization based on rank aggregation. We show that Vaquita is robust in terms of sequencing coverage, insertion size of the library, and read length, and is comparable or even better for the identification of deletions, inversions, duplications, and translocations than state-of-the-art tools, using both simulated and real datasets. In addition, Vaquita is more than eight times faster than any other tools in comparison. Availability: Vaquita is implemented in C++ using the SeqAn library. The source code is distributed under the BSD license and can be downloaded at http://github.com/seqan/vaquita