Andrew Schuster | University of Nevada, Reno (original) (raw)
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Papers by Andrew Schuster
The Journal of biological chemistry, Jan 21, 2014
PIWI-interacting RNAs (piRNAs) are small noncoding RNAs that bind PIWI family proteins exclusivel... more PIWI-interacting RNAs (piRNAs) are small noncoding RNAs that bind PIWI family proteins exclusively expressed in the germ cells of mammalian gonads. MIWI2-associated piRNAs are essential for silencing transposons during primordial germ cell development, and MIWI-bound piRNAs are required for normal spermatogenesis during adulthood in mice. Although piRNAs have long been regarded as germ cell-specific, increasing lines of evidence suggest that somatic cells also express piRNA-like RNAs (pilRNAs). Here, we report the detection of abundant pilRNAs in somatic cells, which are similar to MIWI-associated piRNAs mainly expressed in pachytene spermatocytes and round spermatids in the testis. Based on small RNA deep sequencing and quantitative PCR analyses, pilRNA expression is dynamic and displays tissue specificity. Although pilRNAs are similar to pachytene piRNAs in both size and genomic origins, they have a distinct ping-pong signature. Furthermore, pilRNA biogenesis appears to utilize a ...
Biology of Reproduction, 2013
In mammals, the transcriptome of large noncoding RNAs (lncRNAs) is believed to be greater than th... more In mammals, the transcriptome of large noncoding RNAs (lncRNAs) is believed to be greater than that of messenger RNAs (mRNAs). Some lncRNAs, especially large intergenic noncoding RNAs (lincRNAs), participate in epigenetic regulation by binding chromatin-modifying protein complexes and regulating proteincoding gene expression. Given that epigenetic regulation plays a critical role in male germline development, we embarked on expression profiling of both lncRNAs and mRNAs during male germline reprogramming and postnatal development using microarray analyses. We identified thousands of lncRNAs and hundreds of lincRNAs that are either up-or downregulated at six critical time points during male germ cell development. In addition, highly regulated lncRNAs were correlated with nearby (,30 kb) mRNA gene clusters, which were also significantly upor downregulated. Large ncRNAs can be localized to both the nucleus and cytoplasm, with nuclear lncRNAs mostly associated with key components of the chromatin-remodeling protein complexes. Our data indicate that expression of lncRNAs is dynamically regulated during male germline development and that lncRNAs may function to regulate gene expression at both transcriptional and posttranscriptional levels via genetic and epigenetic mechanisms.
Methods in molecular biology (Clifton, N.J.), 2015
Many classes of small noncoding RNAs (sncRNAs), such as microRNAs (miRNAs) and endogenous small i... more Many classes of small noncoding RNAs (sncRNAs), such as microRNAs (miRNAs) and endogenous small interfering RNAs (endo-siRNAs), have been identified as important regulators of gene expression. Endo-siRNAs represent an integral part of the endogenous RNAi pathway and have been identified in multiple organisms and cell types. Wide adoption of the next-generation deep sequencing (NGS)-based sncRNA profiling has made the identification of novel sncRNA species more accessible. However, it remains a challenge to identify novel endo-siRNAs that are not collected in the current endo-siRNA databases. We have developed an in silico method for identification of novel endo-siRNAs using small RNA NGS data. Here, we describe our protocol in detail.
genesis, 2013
In the Cre-loxp system, expression level and activity of Cre recombinase in a Cre deleter line ar... more In the Cre-loxp system, expression level and activity of Cre recombinase in a Cre deleter line are critical because these determine not only the cell specificity of gene knockout (KO), but also the efficiency of Cre-mediated excision in a specific cell lineage. Although the spatiotemporal expression pattern of a Cre transgene is usually defined upon the generation of the mouse line, the Cre excision efficiency in a specific targeted cell lineage is rarely evaluated and often assumed to be 100%. Incomplete excision can lead to highly variable phenotypes owing to mosaicism (i.e., coexistence of cells with the flox or the recombined flox allele) and this problem has long been overlooked. Here, we report that Stra8-codon-improved Cre recombinase (iCre), a transgenic allele expressing iCre under the control of the male germ cell-specific Stra8 promoter, could efficiently delete one Mov10l1 flox allele in spermatogenic cells, whereas the excision was incomplete when two Mov10l1 flox alleles were present. The incomplete Cre-mediated excision led to a testicular phenotype that was much less severe than that in the true conditional KO (inactivation, 100%) mice. Our findings suggest that it is essential to determine the efficiency of Cre excision when Cre-loxp system is used for deleting genes in a specific cell lineage and the Cre; gene lox/D genotype should be used to evaluate phenotypes instead of Cre; gene lox/lox owing to the fact that the latter usually bears incomplete deletion of the flox allele(s). genesis 00:1-10. V C 2013 Wiley Periodicals, Inc.
Molecular Reproduction and Development, 2013
Cell Death and Differentiation, 2014
The PIWI-piRNA pathway serves as a critical defense mechanism through which the genome of the mal... more The PIWI-piRNA pathway serves as a critical defense mechanism through which the genome of the male germline is protected from invasion by transposable elements (TEs). MIWI2/PIWIL4, a member of the murine PIWI subclade of the Argonaute family, has been shown to be expressed in primordial germ cells (PGCs) and prospermatogonia in fetal and prepubertal testes. Global inactivation of Miwi2 leads to male sterility due to an early meiotic arrest, which correlates with retrotransposon desuppression. However, it remains unclear whether MIWI2 functions beyond the PGC stage and whether MIWI2 has a role beyond TE suppression during male germ line development. Through conditional inactivation of Miwi2, we demonstrate herein that MIWI2 function is restricted to a narrow time window during male PGC reprograming and that Miwi2 is dispensable for postnatal male germline development and testicular function in mice. Moreover, persistent activation of LINE1 and IAP retrotransposons caused by Miwi2 inactivation is compatible with mitotic cell cycle progression of spermatogonia during the first wave of spermatogenesis, but can cause zygotene to pachytene arrest in early meiosis due to multiple defects including enhanced DNA double-strand breaks, aberrant histone modifications and altered mRNA transcriptome. Our data not only validate those from global Miwi2 KO studies, but also suggest that MIWI2 and MIWI2-associated piRNAs have functions beyond TE suppression. Temporally restricted role of MIWI2 in the testis J Bao et al 2 Cell Death and Differentiation Temporally restricted role of MIWI2 in the testis J Bao et al 3 Cell Death and Differentiation Temporally restricted role of MIWI2 in the testis J Bao et al 5 Cell Death and Differentiation Temporally restricted role of MIWI2 in the testis J Bao et al 6 Cell Death and Differentiation Temporally restricted role of MIWI2 in the testis J Bao et al 7 Cell Death and Differentiation Temporally restricted role of MIWI2 in the testis J Bao et al
Scientific reports, 2015
Paramutations result from interactions between two alleles at a single locus, whereby one induces... more Paramutations result from interactions between two alleles at a single locus, whereby one induces a heritable change in the other. Although common in plants, paramutations are rarely studied in animals. Here, we report a new paramutation mouse model, in which the paramutant allele was induced by an insertional mutation and displayed the "white-tail-tip" (WTT) phenotype. The paramutation phenotype could be transmitted across multiple generations, and the breeding scheme (intercrossing vs. outcrossing) drastically affected the transmission efficiency. Paternal (i.e., sperm-borne) RNAs isolated from paramutant mice could induce the paramutation phenotype, which, however, failed to be transmitted to subsequent generations. Maternal miRNAs and piRNAs appeared to have an inhibitory effect on the efficiency of germline transmission of the paramutation. This paramutation mouse model represents an important tool for dissecting the underlying mechanism, which should be applicable to...
The Journal of biological chemistry, Jan 21, 2014
PIWI-interacting RNAs (piRNAs) are small noncoding RNAs that bind PIWI family proteins exclusivel... more PIWI-interacting RNAs (piRNAs) are small noncoding RNAs that bind PIWI family proteins exclusively expressed in the germ cells of mammalian gonads. MIWI2-associated piRNAs are essential for silencing transposons during primordial germ cell development, and MIWI-bound piRNAs are required for normal spermatogenesis during adulthood in mice. Although piRNAs have long been regarded as germ cell-specific, increasing lines of evidence suggest that somatic cells also express piRNA-like RNAs (pilRNAs). Here, we report the detection of abundant pilRNAs in somatic cells, which are similar to MIWI-associated piRNAs mainly expressed in pachytene spermatocytes and round spermatids in the testis. Based on small RNA deep sequencing and quantitative PCR analyses, pilRNA expression is dynamic and displays tissue specificity. Although pilRNAs are similar to pachytene piRNAs in both size and genomic origins, they have a distinct ping-pong signature. Furthermore, pilRNA biogenesis appears to utilize a ...
Biology of Reproduction, 2013
In mammals, the transcriptome of large noncoding RNAs (lncRNAs) is believed to be greater than th... more In mammals, the transcriptome of large noncoding RNAs (lncRNAs) is believed to be greater than that of messenger RNAs (mRNAs). Some lncRNAs, especially large intergenic noncoding RNAs (lincRNAs), participate in epigenetic regulation by binding chromatin-modifying protein complexes and regulating proteincoding gene expression. Given that epigenetic regulation plays a critical role in male germline development, we embarked on expression profiling of both lncRNAs and mRNAs during male germline reprogramming and postnatal development using microarray analyses. We identified thousands of lncRNAs and hundreds of lincRNAs that are either up-or downregulated at six critical time points during male germ cell development. In addition, highly regulated lncRNAs were correlated with nearby (,30 kb) mRNA gene clusters, which were also significantly upor downregulated. Large ncRNAs can be localized to both the nucleus and cytoplasm, with nuclear lncRNAs mostly associated with key components of the chromatin-remodeling protein complexes. Our data indicate that expression of lncRNAs is dynamically regulated during male germline development and that lncRNAs may function to regulate gene expression at both transcriptional and posttranscriptional levels via genetic and epigenetic mechanisms.
Methods in molecular biology (Clifton, N.J.), 2015
Many classes of small noncoding RNAs (sncRNAs), such as microRNAs (miRNAs) and endogenous small i... more Many classes of small noncoding RNAs (sncRNAs), such as microRNAs (miRNAs) and endogenous small interfering RNAs (endo-siRNAs), have been identified as important regulators of gene expression. Endo-siRNAs represent an integral part of the endogenous RNAi pathway and have been identified in multiple organisms and cell types. Wide adoption of the next-generation deep sequencing (NGS)-based sncRNA profiling has made the identification of novel sncRNA species more accessible. However, it remains a challenge to identify novel endo-siRNAs that are not collected in the current endo-siRNA databases. We have developed an in silico method for identification of novel endo-siRNAs using small RNA NGS data. Here, we describe our protocol in detail.
genesis, 2013
In the Cre-loxp system, expression level and activity of Cre recombinase in a Cre deleter line ar... more In the Cre-loxp system, expression level and activity of Cre recombinase in a Cre deleter line are critical because these determine not only the cell specificity of gene knockout (KO), but also the efficiency of Cre-mediated excision in a specific cell lineage. Although the spatiotemporal expression pattern of a Cre transgene is usually defined upon the generation of the mouse line, the Cre excision efficiency in a specific targeted cell lineage is rarely evaluated and often assumed to be 100%. Incomplete excision can lead to highly variable phenotypes owing to mosaicism (i.e., coexistence of cells with the flox or the recombined flox allele) and this problem has long been overlooked. Here, we report that Stra8-codon-improved Cre recombinase (iCre), a transgenic allele expressing iCre under the control of the male germ cell-specific Stra8 promoter, could efficiently delete one Mov10l1 flox allele in spermatogenic cells, whereas the excision was incomplete when two Mov10l1 flox alleles were present. The incomplete Cre-mediated excision led to a testicular phenotype that was much less severe than that in the true conditional KO (inactivation, 100%) mice. Our findings suggest that it is essential to determine the efficiency of Cre excision when Cre-loxp system is used for deleting genes in a specific cell lineage and the Cre; gene lox/D genotype should be used to evaluate phenotypes instead of Cre; gene lox/lox owing to the fact that the latter usually bears incomplete deletion of the flox allele(s). genesis 00:1-10. V C 2013 Wiley Periodicals, Inc.
Molecular Reproduction and Development, 2013
Cell Death and Differentiation, 2014
The PIWI-piRNA pathway serves as a critical defense mechanism through which the genome of the mal... more The PIWI-piRNA pathway serves as a critical defense mechanism through which the genome of the male germline is protected from invasion by transposable elements (TEs). MIWI2/PIWIL4, a member of the murine PIWI subclade of the Argonaute family, has been shown to be expressed in primordial germ cells (PGCs) and prospermatogonia in fetal and prepubertal testes. Global inactivation of Miwi2 leads to male sterility due to an early meiotic arrest, which correlates with retrotransposon desuppression. However, it remains unclear whether MIWI2 functions beyond the PGC stage and whether MIWI2 has a role beyond TE suppression during male germ line development. Through conditional inactivation of Miwi2, we demonstrate herein that MIWI2 function is restricted to a narrow time window during male PGC reprograming and that Miwi2 is dispensable for postnatal male germline development and testicular function in mice. Moreover, persistent activation of LINE1 and IAP retrotransposons caused by Miwi2 inactivation is compatible with mitotic cell cycle progression of spermatogonia during the first wave of spermatogenesis, but can cause zygotene to pachytene arrest in early meiosis due to multiple defects including enhanced DNA double-strand breaks, aberrant histone modifications and altered mRNA transcriptome. Our data not only validate those from global Miwi2 KO studies, but also suggest that MIWI2 and MIWI2-associated piRNAs have functions beyond TE suppression. Temporally restricted role of MIWI2 in the testis J Bao et al 2 Cell Death and Differentiation Temporally restricted role of MIWI2 in the testis J Bao et al 3 Cell Death and Differentiation Temporally restricted role of MIWI2 in the testis J Bao et al 5 Cell Death and Differentiation Temporally restricted role of MIWI2 in the testis J Bao et al 6 Cell Death and Differentiation Temporally restricted role of MIWI2 in the testis J Bao et al 7 Cell Death and Differentiation Temporally restricted role of MIWI2 in the testis J Bao et al
Scientific reports, 2015
Paramutations result from interactions between two alleles at a single locus, whereby one induces... more Paramutations result from interactions between two alleles at a single locus, whereby one induces a heritable change in the other. Although common in plants, paramutations are rarely studied in animals. Here, we report a new paramutation mouse model, in which the paramutant allele was induced by an insertional mutation and displayed the "white-tail-tip" (WTT) phenotype. The paramutation phenotype could be transmitted across multiple generations, and the breeding scheme (intercrossing vs. outcrossing) drastically affected the transmission efficiency. Paternal (i.e., sperm-borne) RNAs isolated from paramutant mice could induce the paramutation phenotype, which, however, failed to be transmitted to subsequent generations. Maternal miRNAs and piRNAs appeared to have an inhibitory effect on the efficiency of germline transmission of the paramutation. This paramutation mouse model represents an important tool for dissecting the underlying mechanism, which should be applicable to...