Heng-Chi Lee

Diverse Pathways Generate MicroRNA-like RNAs and Dicer-Independent Small Interfering RNAs in Fungi

A variety of small RNAs, including the Dicer-dependent miRNAs and the Dicer-independent Piwi-interacting RNAs, associate with Argonaute family proteins to regulate gene expression in diverse cellular processes. These two species of small RNA have not been found in fungi. Here, by analyzing small RNAs associated with the Neurospora Argonaute protein QDE-2, we show that diverse pathways generate miRNA-like small RNAs (milRNAs) and Dicer-independent small interfering RNAs (disiRNAs) in this filamentous fungus. Surprisingly, milRNAs are produced by at least four different mechanisms that use a distinct combination of factors, including Dicers, QDE-2, the exonuclease QIP, and an RNase III domain-containing protein, MRPL3. In contrast, disiRNAs originate from loci producing overlapping sense and antisense transcripts, and do not require the known RNAi components for their production. Taken together, these results uncover several pathways for small RNA production in filamentous fungi, shedding light on the diversity and evolutionary origins of eukaryotic small RNAs.

C. elegans piRNAs Mediate the Genome-wide Surveillance of Germline Transcripts

Piwi Argonautes and Piwi-interacting RNAs (piRNAs) mediate genome defense by targeting transposons. However, many piRNA species lack obvious sequence complementarity to transposons or other loci; only one C. elegans transposon is a known piRNA target. Here, we show that, in mutants lacking the Piwi Argonaute PRG-1 (and consequently its associated piRNAs/21U-RNAs), many silent loci in the germline exhibit increased levels of mRNA expression with a concomitant depletion of RNA-dependent RNA polymerase (RdRP)-derived secondary small RNAs termed 22G-RNAs. Sequences depleted of 22G-RNAs are proximal to potential target sites that base pair imperfectly but extensively to 21U-RNAs. We show that PRG-1 is required to initiate, but not to maintain, silencing of transgenes engineered to contain complementarity to endogenous 21U-RNAs. Our findings support a model in which C. elegans piRNAs utilize their enormous repertoire of targeting capacity to scan the germline transcriptome for foreign sequences, while endogenous germline-expressed genes are actively protected from piRNA-induced silencing.

qiRNA is a new type of small interfering RNA induced by DNA damage

RNA interference pathways use small RNAs to mediate gene silencing in eukaryotes. In addition to small interfering RNAs (siRNAs) and microRNAs, several types of endogenously produced small RNAs have important roles in gene regulation, germ cell maintenance and transposon silencing. The production of some of these RNAs requires the synthesis of aberrant RNAs (aRNAs) or pre-siRNAs, which are specifically recognized by RNA-dependent RNA polymerases to make double-stranded RNA. The mechanism for aRNA synthesis and recognition is largely unknown. Here we show that DNA damage induces the expression of the Argonaute protein QDE-2 and a new class of small RNAs in the filamentous fungus Neurospora crassa. This class of small RNAs, known as qiRNAs because of their interaction with QDE-2, are about 20–21 nucleotides long (several nucleotides shorter than Neurospora siRNAs), with a strong preference for uridine at the 5′ end, and originate mostly from the ribosomal DNA locus. The production of qiRNAs requires the RNA-dependent RNA polymerase QDE-1, the Werner and Bloom RecQ DNA helicase homologue QDE-3 and dicers. qiRNA biogenesis also requires DNA-damage-induced aRNAs as precursors, a process that is dependent on both QDE-1 and QDE-3. Notably, our results suggest that QDE-1 is the DNA-dependent RNA polymerase that produces aRNAs. Furthermore, the Neurospora RNA interference mutants show increased sensitivity to DNA damage, suggesting a role for qiRNAs in the DNA-damage response by inhibiting protein translation.

A Double-Stranded-RNA Response Program Important for RNA Interference Efficiency

ABSTRACT When recognized by the RNA interference (RNAi) pathway, double-stranded RNA (dsRNA) produced in eukaryotic cells results in posttranscriptional gene silencing. In addition, dsRNA can trigger the interferon response as part of the immune response in vertebrates. In this study, we show that dsRNA, but not short interfering RNA (siRNA), induces the expression of qde-2 (an Argonaute gene) and dcl-2 (a Dicer gene), two central components of the RNAi pathway in the filamentous fungus Neurospora crassa. The induction of QDE-2 by dsRNA is required for normal gene silencing, indicating that this is a regulatory mechanism that allows the optimal function of the RNAi pathway. In addition, we demonstrate that Dicer proteins (DCLs) regulate QDE-2 posttranscriptionally, suggesting a role for DCLs or siRNA in QDE-2 accumulation. Finally, a genome-wide search revealed that additional RNAi components and homologs of antiviral and interferon-stimulated genes are also dsRNA-activated genes in Neurospora. Together, our results suggest that the activation of the RNAi components is part of a broad ancient host defense response against viral and transposon infections.

The DNA/RNA-dependent RNA polymerase QDE-1 generates aberrant RNA and dsRNA for RNAi in a process requiring replication protein A and a DNA helicase.

The Neurospora RNA-dependent RNA polymerase QDE-1 is an RNA polymerase that can use both RNA and DNA as templates, suggesting a new mechanism for small RNA production.

The piRNA targeting rules and the resistance to piRNA silencing in endogenous genes

Self-defense by avoiding self-targeting By silencing transposons, Piwi-interacting RNAs (piRNAs) protect the stability of animal genomes in germ lines. However, many piRNAs do not map to transposons, and their functions have remained undefined. Zhang et al. described the piRNA targeting logic in Caenorhabditis elegans and identified an intrinsic sequence signal in endogenous germline genes that confer resistance to piRNA silencing. Thus, diverse piRNAs silence foreign nucleic acids but spare self genes to defend the C. elegans genome. In addition, multiple foreign transgenes can be engineered to escape piRNA targeting, allowing successful expression in the germline. Science, this issue p. 587 In nematodes, endogenous genes are resistant to Piwi-interacting RNA (piRNA) silencing, allowing a role for piRNA in defense against foreign nucleic acids. Piwi-interacting RNAs (piRNAs) silence transposons to safeguard genome integrity in animals. However, the functions of the many piRNAs that do not map to transposons remain unknown. Here, we show that piRNA targeting in Caenorhabditis elegans can tolerate a few mismatches but prefer perfect pairing at the seed region. The broad targeting capacity of piRNAs underlies the germline silencing of transgenes in C. elegans. Transgenes engineered to avoid piRNA recognition are stably expressed. Many endogenous germline-expressed genes also contain predicted piRNA targeting sites, and periodic An/Tn clusters (PATCs) are an intrinsic signal that provides resistance to piRNA silencing. Together, our study revealed the piRNA targeting rules and highlights a distinct strategy that C. elegans uses to distinguish endogenous from foreign nucleic acids.

The Coding Regions of Germline mRNAs Confer Sensitivity to Argonaute Regulation in C. elegans

SUMMARY Protein-coding genes undergo a wide array of regulatory interactions with factors that engage non-coding regions. Open reading frames (ORFs), in contrast, are thought to be constrained by coding function, precluding a major role in gene regulation. Here, we explore Piwi-interacting (pi)RNA-mediated transgene silencing in C. elegans and show that marked differences in the sensitivity to piRNA silencing map to the endogenous sequences within transgene ORFs. Artificially increasing piRNA targeting within the ORF of a resistant transgene can lead to a partial yet stable reduction in expression, revealing that piRNAs not only silence but can also “tune” gene expression. Our findings support a model that involves a temporal element to mRNA regulation by germline Argonautes, likely prior to translation, and suggest that piRNAs afford incremental control of germline mRNA expression by targeting the body of the mRNA, including the coding region.

pirScan: a webserver to predict piRNA targeting sites and to avoid transgene silencing in C. elegans

Abstract pirScan is a web-based tool for identifying C. elegans piRNA-targeting sites within a given mRNA or spliced DNA sequence. The purpose of our tool is to allow C. elegans researchers to predict piRNA targeting sites and to avoid the persistent germline silencing of transgenes that has rendered many constructs unusable. pirScan fulfills this purpose by first enumerating the predicted piRNA-targeting sites present in an input sequence. This prediction can be exported in a tabular or graphical format. Subsequently, pirScan suggests silent mutations that can be introduced to the input sequence that would allow the modified transgene to avoid piRNA targeting. The user can customize the piRNA targeting stringency and the silent mutations that he/she wants to introduce into the sequence. The modified sequences can be re-submitted to be certain that any previously present piRNA-targeting sites are now absent and no new piRNA-targeting sites are accidentally generated. This revised sequence can finally be downloaded as a text file and/or visualized in a graphical format. pirScan is freely available for academic use at http://cosbi4.ee.ncku.edu.tw/pirScan/.

piRTarBase: a database of piRNA targeting sites and their roles in gene regulation

Abstract PIWI-interacting RNAs (piRNAs) are a class of small noncoding RNAs that guard animal genomes against mutation by silencing transposons. In addition, recent studies have reported that piRNAs silence various endogenous genes. Tens of thousands of distinct piRNAs made in animals do not pair well to transposons and currently the functions and targets of piRNAs are largely unexplored. piRTarBase provides a user-friendly interface to access both predicted and experimentally identified piRNA targeting sites in Caenorhabditis elegans. The user can input genes of interest and retrieve a list of piRNA targeting sites on the input genes. Alternatively, the user can input a piRNA and retrieve a list of its mRNA targets. Additionally, piRTarBase integrates published mRNA and small RNA sequencing data, which will help users identify biologically relevant targeting events. Importantly, our analyses suggest that the piRNA sites found by both predictive and experimental approaches are more likely to exhibit silencing effects on their targets than each method alone. Taken together, piRTarBase offers an integrative platform that will help users to identify functional piRNA target sites by evaluating various information. piRTarBase is freely available for academic use at http://cosbi6.ee.ncku.edu.tw/piRTarBase/.