Project description:Antisense oligonucleotides (ASOs) that trigger RNase-H-mediated cleavage are commonly used to knock down transcripts for experimental or therapeutic purposes. In particular, ASOs are frequently used to functionally interrogate long noncoding RNAs (lncRNAs) and discriminate lncRNA loci that produce functional RNAs from those whose activity is attributable to the act of transcription. Transcription termination is triggered by cleavage of nascent transcripts, generally during polyadenylation, resulting in degradation of the residual RNA polymerase II (Pol II)-associated RNA by XRN2 and dissociation of elongating Pol II. Here, we show that ASOs act upon nascent transcripts and, consequently, induce premature transcription termination downstream of the cleavage site in an XRN2-dependent manner. Targeting the transcript 3' end with ASOs, however, allows transcript knockdown while preserving Pol II association with the gene body. These results demonstrate that the effects of ASOs on transcription must be considered for appropriate experimental and therapeutic use of these reagents.
Project description:The Heat Shock response (HSR) is a highly conserved transcriptional program induced by the exposure to a variety of environmental stressors. Following an insult, a small subset of genes, known as the Heat Shock genes, are rapidly induced by the Heat Shock Factors (HSFs) to maintain protein homeostasis and ensure cell survival. In parallel, a large portion of the genome becomes repressed. HS associated global gene repression is still largely poorly understood. In this study, we demonstrate that transcription downregulation during Heat shock is mainly achieved by premature transcription termination through the disruption of telescripting.
Project description:The pervasive nature of RNA polymerase II (Pol II) transcription requires efficient termination. A key player in this process is the cleavage and polyadenylation (CPA) factor PCF11, which directly binds to the Pol II C-terminal domain and dismantles elongating Pol II from DNA in vitro. We demonstrate that PCF11-mediated termination is essential for vertebrate development. A range of genomic analyses, including mNET-seq, 3' mRNA-seq, chromatin RNA-seq, and ChIP-seq, reveals that PCF11 enhances transcription termination and stimulates early polyadenylation genome-wide. PCF11 binds preferentially between closely spaced genes, where it prevents transcriptional interference and consequent gene downregulation. Notably, PCF11 is sub-stoichiometric to the CPA complex. Low levels of PCF11 are maintained by an auto-regulatory mechanism involving premature termination of its own transcript and are important for normal development. Both in human cell culture and during zebrafish development, PCF11 selectively attenuates the expression of other transcriptional regulators by premature CPA and termination.
Project description:RNA Polymerase II (Pol II) termination is dependent on RNA processing signals as well as specific terminator elements located downstream of the poly(A) site. One of the two major terminator classes described so far is the Co-Transcriptional Cleavage (CoTC) element. We show that homopolymer A/T tracts within the human β-globin CoTC-mediated terminator element play a critical role in Pol II termination. These short A/T tracts, dispersed within seemingly random sequences, are strong terminator elements, and bioinformatics analysis confirms the presence of such sequences in 70% of the putative terminator regions (PTRs) genome-wide.
Project description:mRNAs containing premature termination codons (PTCs) are known to be degraded via nonsense-mediated mRNA decay (NMD). Unexpectedly, we found that mRNAs containing any type of PTC (UAA, UAG, UGA) are detained in the nucleus whereas their wild-type counterparts are rapidly exported. This retention is strictly reading-frame dependent. Strikingly, our data indicate that translating ribosomes in the nucleus proofread the frame and detect the PTCs in the nucleus. Moreover, the shuttling NMD protein Upf1 specifically associates with PTC+ mRNA in the nucleus and is required for nuclear retention of PTC+ mRNA. Together, our data lead to a working model that PTCs are recognized in the nucleus by translating ribosomes, resulting in recruitment of Upf1, which in turn functions in nuclear retention of PTC+ mRNA. Nuclear PTC recognition adds a new layer of proofreading for mRNA and may be vital for ensuring the extraordinary fidelity required for protein production.
Project description:Peroxisome abundance is regulated by homeostasis between the peroxisomal biogenesis and degradation processes. Peroxin 16 (PEX16) is a peroxisomal protein involved in trafficking membrane proteins for de novo peroxisome biogenesis. The present study demonstrates that PEX16 also modulates peroxisome abundance through pexophagic degradation. PEX16 knockdown in human retinal pigment epithelial-1 cells decreased peroxisome abundance and function, represented by reductions in the expression of peroxisome membrane protein ABCD3 and the levels of cholesterol and plasmalogens, respectively. The activation of pexophagy under PEX16 knockdown was shown by (i) abrogated peroxisome loss under PEX16 knockdown in autophagy-deficient ATG5 knockout cell lines, and (ii) increased autophagy flux and co-localization of p62-an autophagy adaptor protein-with ABCD3 in the presence of the autophagy inhibitor chloroquine. However, the levels of cholesterol and plasmalogens did not recover despite the restoration of peroxisome abundance following chloroquine treatment. Thus, PEX16 is indispensable for maintaining peroxisome homeostasis by regulating not only the commonly known biogenesis pathway but also the autophagic degradation of peroxisomes.
Project description:Abstract Injection of siRNA (small interfering RNA) into parthenogenetic adult pea aphids (Acyrthosiphon pisum) is shown here to lead to depletion of a target salivary gland transcript. The siRNA was generated from double stranded RNA that covered most of the open reading frame of the transcript, which we have called Coo2. The Coo2 transcript level decreases dramatically over a 3-day period after injection of siRNA. With a lag of 1 to 2 days, the siCoo2-RNA injected insects died, on average 8 days before the death of control insects injected with siRNA for green fluorescent protein. It appears, therefore, that siRNA injections into adults will be a useful tool in studying the roles of individual transcripts in aphid salivary glands and suggests that siCoo2-RNA injections can be a useful positive control in such studies.
Project description:The ZIC transcription factors are key mediators of embryonic development and ZIC3 is the gene most commonly associated with situs defects (heterotaxy) in humans. Half of patient ZIC3 mutations introduce a premature termination codon (PTC). In vivo, PTC-containing transcripts might be targeted for nonsense-mediated decay (NMD). NMD efficiency is known to vary greatly between transcripts, tissues and individuals and it is possible that differences in survival of PTC-containing transcripts partially explain the striking phenotypic variability that characterizes ZIC3-associated congenital defects. For example, the PTC-containing transcripts might encode a C-terminally truncated protein that retains partial function or that dominantly interferes with other ZIC family members. Here we describe the katun (Ka) mouse mutant, which harbours a mutation in the Zic3 gene that results in a PTC. At the time of axis formation there is no discernible decrease in this PTC-containing transcript in vivo, indicating that the mammalian Zic3 transcript is relatively insensitive to NMD, prompting the need to re-examine the molecular function of the truncated proteins predicted from human studies and to determine whether the N-terminal portion of ZIC3 possesses dominant-negative capabilities. A combination of in vitro studies and analysis of the Ka phenotype indicate that it is a null allele of Zic3 and that the N-terminal portion of ZIC3 does not encode a dominant-negative molecule. Heterotaxy in patients with PTC-containing ZIC3 transcripts probably arises due to loss of ZIC3 function alone.
Project description:Rapid and reversible methods for altering the levels of endogenous proteins are critically important for studying biological systems and developing therapeutics. Here we describe a membrane-permeant targeting peptide-based method that rapidly and reversibly knocks down endogenous proteins through chaperone-mediated autophagy in vitro and in vivo. We demonstrate the specificity, efficacy and generalizability of the method by showing efficient knockdown of various proteins, including death associated protein kinase 1 (160 kDa), scaffolding protein PSD-95 (95 kDa) and α-synuclein (18 kDa), with their respective targeting peptides in a dose-, time- and lysosomal activity-dependent manner in rat neuronal cultures. Moreover, we show that, when given systemically, the peptide system efficiently knocked down the targeted protein in the brains of intact rats. Our study provides a robust and convenient research tool for manipulating endogenous protein levels and may also lead to the development of protein knockdown-based therapeutics for treating human diseases.