key: cord-0327681-ak8l7aet authors: Tsai, Kevin; Bogerd, Hal P.; Kennedy, Edward M.; Emery, Ann; Swanstrom, Ronald; Cullen, Bryan R. title: Epitranscriptomic addition of m6A regulates HIV-1 RNA stability and alternative splicing date: 2021-02-23 journal: bioRxiv DOI: 10.1101/2021.02.23.432449 sha: fb80492e7579cd6543255e97713d52e67da781e4 doc_id: 327681 cord_uid: ak8l7aet Previous work in several laboratories has demonstrated that the epitranscriptomic addition of m6A to viral transcripts promotes the replication and pathogenicity of a wide range of DNA and RNA viruses, yet the underlying mechanisms responsible for this positive effect have remained unclear. It is known that m6A function is largely mediated by cellular m6A binding proteins or readers, yet how m6A readers regulate viral gene expression in general, and HIV-1 gene expression in particular, has been controversial. Here, we confirm that m6A addition indeed regulates HIV-1 RNA expression and demonstrate that this effect is in large part mediated by the the nuclear m6A reader YTHDC1 and the cytoplasmic m6A reader YTHDF2. Both YTHDC1 and YTHDF2 bind to multiple distinct and overlapping sites on the HIV-1 RNA genome, with YTHDC1 recruitment serving to regulate the alternative splicing of HIV-1 RNAs while YTHDF2 binding correlates with increased HIV-1 transcript stability. Author Summary This manuscript reports that the expression of mRNAs encoded by the pathogenic human retrovirus HIV-1 is regulated by the methylation of a small number of specific adenosine residues. These in turn recruit a nuclear RNA binding protein, called YTHDC1, which modulates the alternative splicing of HIV-1 transcripts, as well as a cytoplasmic RNA binding protein, called YTHDF2, which stabilizes viral mRNAs. The regulation of HIV-1 gene expression by adenosine methylation is therefore critical for the effective and ordered expression of HIV-1 mRNAs and could represent a novel target for antiviral development. Eukaryotic mRNAs are subject to a range of covalent modifications at the 46 nucleotide level, collectively referred to as epitranscriptomic modifications. The most 47 common epitranscriptomic modification of mammalian mRNAs is methylation of the N 6 48 position of adenosine, referred to as m 6 A, which comprises ~0.4% of all adenosines [1, 2] . 49 m 6 A residues are deposited on mRNAs by a "writer" complex, minimally consisting of 50 METTL3, METTL14 and WTAP, which adds m 6 A to some, but not all, copies of the RNA 51 motif 5'-RRACH-3' (R=G/A, H=A/U/C) [3] [4] [5] [6] . Cells also express two demethylases or 52 "erasers", called FTO and ALKBH5, which have been proposed to dynamically regulate 53 m 6 A levels [7, 8] . Finally, the phenotypic effects of m 6 A on mRNA metabolism are largely 54 conferred by m 6 A binding proteins, referred to as m 6 A readers, that regulate several steps 55 of RNA metabolism. These include the nuclear m 6 A reader YTHDC1, which has been 56 reported to regulate cellular mRNA splicing and nuclear export, and the cytoplasmic m 6 A 57 readers YTHDF1, YTHDF2 and YTHDF3, which are thought to regulate the translation 58 and stability of m 6 A-containing mRNAs [9] [10] [11] [12] . Recent reports suggest that the cytosolic 59 readers YTHDF1, 2 and 3 confer redundant functions, with YTHDF2 the dominant reader 60 due to its higher expression level [13, 14] . 61 While m 6 A is the most common epitranscriptomic modification on cellular 62 mRNAs, analysis of the genomic RNAs encoded by retroviruses revealed an even higher 63 level of m 6 A on these transcripts, with m 6 A comprising as much as ~2.4% of the 64 adenosines found on murine leukemia virus (MLV) genomic RNAs [15] . This high 65 prevalence suggests that m 6 A might be acting to promote some aspect(s) of the viral 66 replication cycle. Indeed, addition of m 6 A has now been shown to boost the replication of 67 spliced or incompletely spliced HIV-1 RNAs, we isolated nuclear or cytoplasmic fractions 228 from WT or YTHDC1 knock down 293T cells. Analysis of these fractions by Western 229 analysis for the nuclear protein Lamin or the cytoplasmic protein GAPDH confirmed the 230 integrity of the fractionation process and further confirmed not only the nuclear localization 231 of YTHDC1 but also the effective knock down of YTHDC1 protein expression by RNAi 232 Primer-ID-based deep sequencing of splice forms [31] to quantitatively measure the effect 247 of YTHDC1 on HIV-1 alternative splicing. In this assay, a common forward primer that 248 anneals 5' to the gag gene can be paired with either random reverse primers, or splice form-249 specific 4kb or 1.8kb reverse primers, to selectively amplify viral transcripts (Fig. 6A ). HIV-1 transcripts are divided by size into the ~9kb unspliced transcript, ~4kb incompletely 251 spliced transcripts (which retain the D4/A7 env intron), and ~1.8kb fully spliced transcripts, 252 (lacking the D4/A7 intron). While the random reverse primer amplifies all viral transcripts, 253 the 4kb reverse primer, located within the D4/A7 intron, only amplifies incompletely 254 spliced transcripts. The 1.8kb reverse primer spans the D4/A7 splice junction and thus only 255 amplifies fully spliced transcripts. Using the random reverse primer, we first noted that 256 ~33% of viral transcripts are spliced in the siCtrl cells, while this increased to ~42% in the 257 siDC1 cells but decreased to ~25% in the +DC1 cells (Fig. 6B ). Yet, among spliced 258 transcripts, a constant ~85% of all spliced transcripts were fully spliced, that is lacking the 259 D4/A7 intron, regardless of YTHDC1 expression level (Fig. 6C ). Thus, YTHDC1 has a 260 stronger inhibitory effect on utilization of splice donor D1 than D4. Quantification of the 261 level of splicing between donor D1 and the central splice acceptors A1-A5 (Fig. 6D) , using 262 the random reverse primer, revealed that YTHDC1 overexpression led to a significant bias 263 towards utilization of the more 3' acceptors A3, A4 and A5, at the expense of the more 5' 264 acceptors A1 and A2 (Fig. 6D ) and, as expected, the opposite result was observed in cells 265 in which YTHDC1 expression was knocked down, ie. increased utilization of A1 and A2 266 and a concomitant reduction in the utilization of A3, A4 and A5. The class-specific 1.8 and 267 4kb reverse primers confirmed this bias (Figs. 6E and F). YTHDC1 over-expression also 268 led to a significant, ~2.5x increase in the detection of the normally very rare D1 to A7 269 splice (Fig. 6G) , consistent with the observed bias towards splicing D1 to more 3' splice 270 acceptors in YTHDC1 overexpressing cells. Finally, we noted that among the transcripts 271 that used splice acceptor A2, YTHDC1 significantly decreased the proportion of viral 272 the small non-coding exon located between A2 and D3 (Fig. 6A) HIV-1 virions, then they are effectively inactivated due to degradation by the viral protease 307 [23]. However, we were unable to detect the virion packaging of any YTHDF reader 308 protein even when viral protease activity was inhibited (Fig. 2D) . 309 In this report, we have sought to define the precise step(s) in the HIV-1 replication 310 cycle that is regulated by m 6 A-bound reader proteins. In the case of YTHDF2, we show 311 that this reader primarily acts to increase HIV-1 RNA expression, and not translation, and 312 demonstrate that this effect is mediated by enhanced viral RNA stability ( Fig. 2 and 3 . We also note that m 6 A is known to promote the expression 318 of mRNAs encoded by a wide range of viruses [16] , which is clearly inconsistent with the 319 idea that the recruitment of cytoplasmic readers inhibits mRNA expression. Why 320 YTHDF2, and potentially the other cytoplasmic m 6 A readers, stabilize m 6 A-containing 321 viral RNAs yet destabilize most m 6 A-containing cellular RNAs is currently unknown, 322 though it appears possible that this is dependent on the simultaneous recruitment of other 323 RNA binding proteins that remain to be identified, perhaps to adjacent sites on the viral 324 RNA. We note that the inhibitory effect on HIV-1 gene expression in infected T cells seen 325 when expression of the YTHDF2 reader is blocked (Fig. 3) is indistinguishable from the 326 inhibitory effect seen when removal of m 6 A residues from viral transcripts is enhanced by 327 overexpression of the m 6 A eraser ALKBH5 ( Fig. 1 A4 and A5, an effect that was most clearly seen when the random reverse primer splicing 366 assay was used (Fig. 6D) . 367 Previous work looking at the effect of YTHDC1 on splicing of cellular mRNAs 368 largely looked at the effect of YTHDC1 on the inclusion or skipping of cassette exons and 369 concluded that YTHDC1 promoted exon inclusion by recruiting the pre-mRNA splicing 370 factor SRSF3 [10]. However, this report did not address whether the location of m 6 A 371 addition sites relative to splice sites was important. As alternative splicing of HIV-1 RNAs 372 is a much more complex process than simple exon inclusion or exclusion, it is hard to 373 address whether our data are consistent with this earlier report. However, we note that none 374 of the mapped YTHDC1 binding sites on the HIV-1 genome colocalize with either known 375 viral splicing enhancer/repressor elements or with any intronic branch points (consensus 376 motif 5'-YNYURAY-3') [41]. A minor peak of YTHDC1 binding does precisely overlap 377 with splice acceptor A7, with the only m 6 A motif present in this peak (5'-RRm 6 ACH-3') 378 positioning the splice junction exactly 5' to the m 6 A (Fig. 4B) . YTHDC1 overexpression 379 also biases D1-originating splices away from acceptors A1 and A2, and towards A3, A4, 380 A5 and A7 (Fig. 6 ) and we mapped a YTHDC1 binding site between A2 and A3 (Fig. 4A) , 381 which demarcates the border of this effect. However, determining whether the precise 382 location of the YTHDC1 binding sites mapped on the HIV-1 genome, relative to splice 383 sites, is indeed important may be challenging, given the difficulty of generating silent 384 mutations at many of these sites. The pre-diluted siRNA and Lipofectamine solutions were combined and, 5mins later, 502 added drop-wise to the cells in 6 well plates. 6-8hrs post siRNA transfection, the 503 transfection media was removed from 6 well plates, and the cells overlaid with fresh 504 DMEM. The cells in 6 well plates were subsequently co-transfected with 0.5µg pCMV-505 CD4, 1.5µg pBC-CXCR4, and 1µg of either empty pK vector or pK-FLAG-YTHDC1, 506 using PEI. On day 2, the media of 10cm and 6 well plates were exchanged for fresh DMEM, 507 and the 6 well plates were subject to a second round of siRNA transfection. The 508 transfections were coordinated for a control well transfected with pK vector + siCtrl, a 509 knockdown well of pK vector + siDC1 and an overexpression well of pK-FLAG-YTHDC1 510 + siCtrl. On day 3, the media was changed again on the 6 well plates to remove the 511 transfection mix. Day 4, the cells in the 6 well plates were trypsinized, counted and seeded 512 Script III (invitrogen) and random hexamer primers following manufacturer instructions in 559 a 20µl reaction at 50°C 1hr followed by 15mins inactivation at 70°C. Each 20µl RT 560 reaction was diluted with dH2O to a final 120µl. qPCR was performed with 6µl diluted RT 561 product, 8µl 2x Power SYBR Green Master Mix (ABI), and 1µl each of 1mM forward and 562 reverse qPCR primer in a 16µl reaction. qPCR readouts were normalized to GAPDH levels 563 using the delta-delta Ct (ΔΔCt) method. All PCR primers used are listed in Supplemental 564 Table 1 . The next day, the virus production plate media was changed, the CD4/CXCR4/FLAG-586 tagged gene of interest transfected plates were split 1 in 5 to a total of 10x 15cm plates of 587 infection target cells. 3 days post transfection, the supernatant of virus production cells 588 were spun 3000rpm 10mins and filtered through 0.45µm pore filters to remove floating 589 cell, diluted 2x with fresh DMEM, and used to infect the infection target plates, resulting 590 in 15ml media/virus mix per plate. 48hpi, 8ml of supernatant were removed from each 591 plate, and replaced with 10ml of fresh media supplemented with 4-thiouridine (4SU) to a 592 final concentration of 100µM. The next day, 16hrs post 4SU pulse, the supernatents were 593 replaced with 3ml/plate of ice cold PBS, and subject to 2500 x100µJ/cm2 of 365nm UV 594 irradiation twice. Cells were then washed twice with PBS, scrapped off plates, and frozen 595 at -80°C. 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Statistical analysis used Student's T test, error bars=SD *<0.05, **<0 expressing the irrelevant GFP-targeted Cas9 (ΔG) ). Also analyzed were ALKBH5 (the 883 overexpressed ALKBH5 is epitope tagged and runs slightly slower than endogenous 884 ALKBH5) and GAPDH, as a loading control.