Small RNAs such as small interfering RNAs (siRNAs) and microRNAs (miRNAs) silence the expression of their complementary target messenger RNAs via the formation of effector RNA-induced silencing complexes (RISCs), which contain Argonaute (Ago) family proteins at their core. Although loading of siRNA duplexes into Drosophila Ago2 requires the Dicer-2-R2D2 heterodimer and the Hsc70/Hsp90 (Hsp90 also known as Hsp83) chaperone machinery, the details of RISC assembly remain unclear. Here we reconstitute RISC assembly using only Ago2, Dicer-2, R2D2, Hsc70, Hsp90, Hop, Droj2 (an Hsp40 homologue) and p23. By following the assembly of single RISC molecules, we find that, in the absence of the chaperone machinery, an siRNA bound to Dicer-2-R2D2 associates with Ago2 only transiently. The chaperone machinery extends the dwell time of the Dicer-2-R2D2-siRNA complex on Ago2, in a manner dependent on recognition of the 5'-phosphate on the siRNA guide strand. We propose that the chaperone machinery supports a productive state of Ago2, allowing it to load siRNA duplexes from Dicer-2-R2D2 and thereby assemble RISC.
This volume focuses on cytological, biochemical, and molecular biological methods to identify and examine the function of each nuclear body, with an emphasis on the analysis of long non-coding RNAs. Chapters focus on exploring recent studies that reveal how certain long non protein-coding RNAs accumulate in specific nuclear bodies and regulate the function of the bodies by serving as architectural components or controlling the dynamics of associating protein components. Written in the highly successful Methods of Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and key tips on troubleshooting and avoiding known pitfalls.
Authoritative and practical, Nuclear Bodies and Noncoding RNAs: Methods and Protocols serves as a guideline for further study into the sophisticated regulation of gene expression.
Paraspeckles are subnuclear structures that form around nuclear paraspeckle assembly transcript 1 (NEAT1) long noncoding RNA (lncRNA). Recently, paraspeckles were shown to be functional nuclear bodies involved in stress responses and the development of specific organs. Paraspeckle formation is initiated by transcription of the NEAT1 chromosomal locus and proceeds in conjunction with NEAT1 lncRNA biogenesis and a subsequent assembly step involving >40 paraspeckle proteins (PSPs). In this study, subunits of SWItch/Sucrose NonFermentable (SWI/SNF) chromatin-remodeling complexes were identified as paraspeckle components that interact with PSPs and NEAT1 lncRNA. EM observations revealed that SWI/SNF complexes were enriched in paraspeckle subdomains depleted of chromatin. Knockdown of SWI/SNF components resulted in paraspeckle disintegration, but mutation of the ATPase domain of the catalytic subunit BRG1 did not affect paraspeckle integrity, indicating that the essential role of SWI/SNF complexes in paraspeckle formation does not require their canonical activity. Knock- down of SWI/SNF complexes barely affected the levels of known essential paraspeckle components, but markedly diminished the interactions between essential PSPs, suggesting that SWI/SNF complexes facilitate organization of the PSP interaction network required for intact paraspeckle assembly. The interactions between SWI/SNF components and essential PSPs were maintained in NEAT1-depleted cells, suggesting that SWI/SNF complexes not only facilitate interactions between PSPs, but also recruit PSPs during paraspeckle assembly. SWI/SNF complexes were also required for Satellite III lncRNA-dependent formation of nuclear stress bodies under heat-shock conditions. Our data suggest the existence of a common mechanism underlying the formation of lncRNA-dependent nuclear body architectures in mammalian cells.
Neat1 is a non-protein-coding RNA that serves as an architectural component of the nuclear bodies known as paraspeckles. Although cell-based studies indicate that Neat1 is a crucial regulator of gene expression, its physiological relevance remains unclear. Here, we find that Neat1 knockout (KO) mice stochastically fail to become pregnant despite normal ovulation. Unilateral transplantation of wild-type ovaries or the administration of progesterone partially rescued the phenotype, suggesting that corpus luteum dysfunction and concomitant low progesterone were the primary causes of the decreased fertility. In contrast to the faint expression observed in most of the adult tissues, Neat1 was highly expressed in the corpus luteum, and the formation of luteal tissue was severely impaired in nearly half of the Neat1 KO mice. These observations suggest that Neat1 is essential for the formation of the corpus luteum and for the subsequent establishment of pregnancy under a suboptimal condition that has not yet been identified.
PIWI-interacting RNAs (piRNAs) silence transposons in animal germ cells. PIWI proteins bind and amplify piRNAs via the "Ping-Pong" pathway. Because PIWI proteins cleave RNAs between target nucleotides t10 and t11-the nucleotides paired to piRNA guide positions g10 and g11-the first ten nucleotides of piRNAs participating in the Ping-Pong amplification cycle are complementary. Drosophila piRNAs bound to the PIWI protein Aubergine typically begin with uridine (1U), while piRNAs bound to Argonaute3, which are produced by Ping-Pong amplification, often have adenine at position 10 (10A). The Ping-Pong model proposes that the 10A is a consequence of 1U. We find that 10A is not caused by 1U. Instead, fly Aubergine as well as its homologs, Siwi in silkmoth and MILI in mice, have an intrinsic preference for adenine at the t1 position of their target RNAs; during Ping-Pong amplification, this t1A subsequently becomes the g10A of a piRNA bound to Argonaute3.
miRNAs silence their complementary target mRNAs by translational repression as well as by poly(A) shortening and mRNA decay. In Drosophila, miRNAs are typically incorporated into Argonaute1 (Ago1) to form the effector complex called RNA-induced silencing complex (RISC). Ago1-RISC associates with a scaffold protein GW182, which recruits additional silencing factors. We have previously shown that miRNAs repress translation initiation by blocking formation of the 48S and 80S ribosomal complexes. However, it remains unclear how ribosome recruitment is impeded. Here, we examined the assembly of translation initiation factors on the target mRNA under repression. We show that Ago1-RISC induces dissociation of eIF4A, a DEAD-box RNA helicase, from the target mRNA without affecting 5' cap recognition by eIF4E in a manner independent of GW182. In contrast, direct tethering of GW182 promotes dissociation of both eIF4E and eIF4A. We propose that miRNAs act to block the assembly of the eIF4F complex during translation initiation.
Although recent transcriptome analyses have uncovered numerous non-coding RNAs (ncRNAs), their functions remain largely unknown. ncRNAs assemble with proteins and operate as ribonucleoprotein (RNP) machineries, formation of which is thought to be determined by specific fundamental elements embedded in the primary RNA transcripts. Knowledge about the relationships between RNA elements, RNP machinery, and molecular and physiological functions is critical for understanding the diverse roles of ncRNAs and may eventually allow their systematic classification or "taxonomy." In this review, we catalog and discuss representative small and long non-coding RNA classes, focusing on their currently known (and unknown) RNA elements and RNP machineries.