In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes.

For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure flux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy.

Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation, it is imperative to target by gene knockout or RNA interference more than one autophagy-related protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular assays, we hope to encourage technical innovation in the field.

/pdf/guidelines-for-the-use-and-interpretation-of-assays-for-ijf2t30pbq.pdf

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

Plant endogenous non-coding short small RNAs (20–24 nt), including microRNAs (miRNAs) and a subset of small interfering RNAs (ta-siRNAs), play important role in gene expression regulatory networks (GRNs). For example, many transcription factors and development-related genes have been reported as targets of these regulatory small RNAs. Although a number of miRNA target prediction algorithms and programs have been developed, most of them were designed for animal miRNAs which are significantly different from plant miRNAs in the target recognition process. These differences demand the development of separate plant miRNA (and ta-siRNA) target analysis tool(s). We present psRNATarget, a plant small RNA target analysis server, which features two important analysis functions: (i) reverse complementary matching between small RNA and target transcript using a proven scoring schema, and (ii) target-site accessibility evaluation by calculating unpaired energy (UPE) required to ‘open’ secondary structure around small RNA’s target site on mRNA. The psRNATarget incorporates recent discoveries in plant miRNA target recognition, e.g. it distinguishes translational and post-transcriptional inhibition, and it reports the number of small RNA/target site pairs that may affect small RNA binding activity to target transcript. The psRNATarget server is designed for high-throughput analysis of next-generation data with an efficient distributed computing back-end pipeline that runs on a Linux cluster. The server front-end integrates three simplified user-friendly interfaces to accept user-submitted or preloaded small RNAs and transcript sequences; and outputs a comprehensive list of small RNA/target pairs along with the online tools for batch downloading, key word searching and results sorting. The psRNATarget server is freely available at http://plantgrn.noble.org/psRNATarget/.

psRNATarget: a plant small RNA target analysis server

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

MicroRNAs (miRNAs) are endogenous ∼23 nt RNAs that play important gene-regulatory roles in animals and plants by pairing to the mRNAs of protein-coding genes to direct their posttranscriptional repression. This review outlines the current understanding of miRNA target recognition in animals and discusses the widespread impact of miRNAs on both the expression and evolution of protein-coding genes.

MicroRNAs: Target Recognition and Regulatory Functions

microRNAs (miRNAs) are small endogenous non-coding RNAs that function as the universal specificity factors in post-transcriptional gene silencing. Discovering miRNAs, identifying their targets and further inferring miRNA functions have been a critical strategy for understanding normal biological processes of miRNAs and their roles in the development of disease. In this review, we focus on computational methods of inferring miRNA functions, including miRNA functional annotation and inferring miRNA regulatory modules, by integrating heterogeneous data sources. We also briefly introduce the research in miRNA discovery and miRNA-target identification with an emphasis on the challenges to computational biology.

/pdf/identifying-mirnas-targets-and-functions-291rp468ms.pdf

Identifying miRNAs, targets and functions

MicroRNAs (miRNAs) are one class of tiny, endogenous RNAs that can regulate messenger RNA (mRNA) expression by targeting homologous sequences in mRNAs. Their aberrant expressions have been observed in many cancers and several miRNAs have been convincingly shown to play important roles in carcinogenesis. Since the discovery of this small regulator, computational methods have been indispensable tools in miRNA gene finding and functional studies. In this review we first briefly outline the biological findings of miRNA genes, such as genomic feature, biogenesis, gene structure, and functional mechanism. We then discuss in detail the three main aspects of miRNA computational studies: miRNA gene finding, miRNA target prediction, and regulation of miRNA genes. Finally, we provide perspectives on some emerging issues, including combinatorial regulation by miRNAs and functional binding sites beyond the 3′-untranslated region (3′UTR) of target mRNAs. Available online resources for miRNA computational studies are also provided.

Computational approaches for microRNA studies: a review.

Macroautophagy (hereafter referred to as autophagy) is an evolutionarily conserved self-degradative process, which involves the regular turnover of cellular components via sequestering damaged macromolecules and transporting them for lysosomal degradation. In the past few years, the scientific community has produced remarkable advances in our understanding of the genes that are involved in autophagy and of their profound effects on various diseases. Recently, a new class of noncoding RNAs, known as microRNAs (miRNAs), has been demonstrated to play crucial roles in diverse biological processes including development, cell differentiation and apoptosis. Here, we review the current understanding about miRNAs focusing on their involvement in the autophagy process. Intriguingly, several confirmed targets of these autophagy-miRNAs are also important regulators in the crosstalk between autophagy and apoptosis. Furthermore, transcripts involved in autophagy and apoptosis may indirectly modulate each other by competing for common miRNA binding sites. Thus, miRNAs potentially work as molecular switches between these two intimately connected processes and contribute to the cell fate decision.

https://www.tandfonline.com/doi/pdf/10.4161/auto.19629

MicroRNAs in autophagy and their emerging roles in crosstalk with apoptosis

Purpose: Cataracts can be broadly divided into two types: congenital cataracts and age-related cataracts. ΔG91 is a previously discovered congenital mutation in βA3-crystallin that impairs protein solubility. On the other hand, the deamidation of β-crystallin is a significant feature in aged and cataractous lenses. Several deamidation sites were also identified in βA3-crystallin. The present study is to compare the functional consequence of ΔG91 mutation and the deamidation of βA3-crystallin in terms of folding properties and protein–protein interaction. Methods: Protein secondary structure and hydrophobic properties were investigated by in silica analysis of the wild type and mutants sequences. Full-length βA3-crystallin was cloned into a mammalian two-hybrid system in order to investigate protein–protein interactions. Deletion and deamidation were introduced by site-directed mutagenesis protocols. Both the Q85 and Q180 deamidation sites were substituted with glutamic acid residues to mimic deamidation. Different combinations of plasmid constructs were transfected in HeLa cells, and changes of protein–protein interactions were analyzed by the luciferase assay. Results: Bioinformatics prediction suggested that ΔG91 mutation alters both the predicted secondary structure and hydrophobic character of βA3-crystallin, while deamidation only exhibits minimal effects. Mammalian two-hybrid results indicated that both ΔG91 mutation and Q85/Q180 deamidation could significantly decrease the interaction of the βA3crystallin homodimer. Conclusion: Our results provided evidence that both mutations involved in congenital cataracts and deamidation in aged lenses commonly altered protein–protein interaction between human lens βA3-crystallins, which may lead to protein insolubilization and contribute to cataracts.

/pdf/decreasing-the-homodimer-interaction-a-common-mechanism-2pwa0rtru2.pdf

Xiaorong Tan

Papers

Computational approaches for microRNA studies: a review.

MicroRNAs in autophagy and their emerging roles in crosstalk with apoptosis

Decreasing the homodimer interaction: a common mechanism shared by the ΔG91 mutation and deamidation in βA3-crystallin