Although thousands of long noncoding RNAs (lncRNAs) have been discovered, very little is known about their mode of action. Here we functionally characterize an E2F1-regulated lncRNA named Khps1, which is transcribed in antisense orientation to the proto-oncogene SPHK1. Khps1 activates SPHK1 expression by recruiting the histone acetyltransferase p300/CBP to the SPHK1 promoter, which leads to local changes of the chromatin structure that ensures E2F1 binding and enhances transcription. Mechanistically, this is achieved by direct association of Khps1 with a homopurine stretch upstream of the transcription start site of SPHK1, which forms a DNA-RNA triplex that anchors the lncRNA and associated effector proteins to the gene promoter. The results reveal an lncRNA- and E2F1-driven regulatory loop in which E2F1-dependent induction of antisense RNA leads to changes in chromatin structure, facilitating E2F1-dependent expression of SPHK1 and restriction of E2F1-induced apoptosis.

BackgroundCells have evolved quality control mechanisms to ensure protein homeostasis by detecting and degrading aberrant mRNAs and proteins. A common source of aberrant mRNAs is premature polyadenylation, which can result in non-functional protein products. Translating ribosomes that encounter poly(A) sequences are terminally stalled, followed by ribosome recycling and decay of the truncated nascent polypeptide via ribosome-associated quality control.ResultsHere, we demonstrate that the conserved RNA-binding E3 ubiquitin ligase Makorin Ring Finger Protein 1 (MKRN1) promotes ribosome stalling at poly(A) sequences during ribosome-associated quality control. We show that MKRN1 directly binds to the cytoplasmic poly(A)-binding protein (PABPC1) and associates with polysomes. MKRN1 is positioned upstream of poly(A) tails in mRNAs in a PABPC1-dependent manner. Ubiquitin remnant profiling and in vitro ubiquitylation assays uncover PABPC1 and ribosomal protein RPS10 as direct ubiquitylation substrates of MKRN1.ConclusionsWe propose that MKRN1 mediates the recognition of poly(A) tails to prevent the production of erroneous proteins from prematurely polyadenylated transcripts, thereby maintaining proteome integrity.

Makorins are evolutionary conserved proteins that contain C 3 H-type zinc finger modules and a RING E3 ubiquitin ligase domain. In Drosophila, maternal Makorin 1 (Mkrn1) has been linked to embryonic patterning but the mechanism remained unsolved. Here, we show that Mkrn1 is essential for axis specification and pole plasm assembly by translational activation of oskar (osk). We demonstrate that Mkrn1 interacts with poly(A) binding protein (pAbp) and binds specifically to osk 3' UTR in a region adjacent to A-rich sequences. Using Drosophila S2R+ cultured cells we show that this binding site overlaps with a Bruno1 (Bru1) responsive element (BREs) that regulates osk translation. We observe increased association of the translational repressor Bru1 with osk mRNA upon depletion of Mkrn1, indicating that both proteins compete for osk binding. Consistently, reducing Bru1 dosage partially rescues viability and Osk protein level in ovaries from Mkrn1 females. We conclude that Mkrn1 controls embryonic patterning and germ cell formation by specifically activating osk translation, most likely by competing with Bru1 to bind to osk 3' UTR.

1Cells have evolved quality control mechanisms to ensure protein homeostasis by 2 detecting and degrading aberrant mRNAs and proteins. A common source of aberrant 3 mRNAs is premature polyadenylation, which can result in non-functional protein 4 products. Translating ribosomes encountering poly(A) sequences are terminally 5 stalled, followed by ribosome recycling and decay of the truncated nascent polypeptide 6 via the ribosome-associated quality control (RQC). Here, we demonstrate that the 7 conserved RNA-binding E3 ubiquitin ligase Makorin Ring Finger Protein 1 (MKRN1) 8 promotes ribosome stalling at poly(A) sequences during RQC. We show that MKRN1 9 is positioned upstream of A-rich stretches and poly(A) tails in mRNAs through an 10 interaction with the cytoplasmic poly(A)-binding protein (PABP). We uncover PABP, 11 ribosomal protein RPS10, and additional translational regulators as main ubiquitylation 12 substrates of MKRN1. Consequently, we propose that MKRN1 serves as a first line of 13 poly(A) recognition at the mRNA level to prevent production of erroneous proteins, thus 14 maintaining proteome integrity. 15 Keywords 16 MKRN1, ubiquitylation, RNA binding, ribosome-associated quality control, RQC, 17 poly(A), iCLIP, ubiquitin remnant profiling, translation 18 PABP-interacting motif 2 (PAM2 motif) in rat neurons (Miroci et al. 2012). 61 Nevertheless, the RNA binding specificity and functional role of MKRN1 in human cells 62 remained largely elusive. 63Here, we introduce MKRN1 as a novel factor in RQC. MKRN1 is recruited to A-rich 64 sequences in mRNAs in a PABP-dependent manner, where it acts as a first line of 65 defence against poly(A) translation. MKRN1 depletion abrogates ribosome stalling in 66 reporter assays, accompanied by reduced ubiquitylation of RQC-related proteins. We 67