The 5´-leader of the HIV-1 genome regulates multiple functions during viral replication by mechanisms that have yet to be established. We developed an NMR approach that enabled direct detection of structural elements within the intact leader (712 nucleotide dimer) that are critical for genome packaging. Residues spanning the gag start codon (AUG) form a hairpin in the monomeric leader and base pair with residues of the Unique-5´ region (U5) in the dimer. U5:AUG formation promotes dimerization by displacing and exposing a dimer-promoting hairpin, and enhances binding by the nucleocapsid protein (NC), the cognate domain of the viral Gag polyprotein that directs packaging. Our findings support a packaging mechanism in which translation, dimerization, NC binding, and packaging are regulated by a common RNA structural switch.

Assembly of Human Immunodeficiency Virus Type-1 (HIV-1) particles is initiated in the cytoplasm by the formation of a ribonucleoprotein complex comprising the dimeric RNA genome and a small number of viral Gag polyproteins. Genomes are recognized by the nucleocapsid (NC) domains of Gag, which interact with packaging elements believed to be located primarily within the 5´-leader of the viral RNA. Recent studies revealed that the native 5´-leader exists as an equilibrium of two conformers, one in which dimer-promoting residues and NC binding sites are sequestered and packaging is attenuated, and one in which these sites are exposed and packaging is promoted. To identify the elements within the dimeric 5´-leader that are important for packaging, we generated HIV-1 5´-leader RNAs containing mutations and deletions designed to eliminate substructures without perturbing the overall structure of the leader and examined effects of the mutations on RNA dimerization, NC binding and packaging. Our findings identify a 159 residue RNA packaging signal that possesses dimerization and NC binding properties similar to those of the intact 5´-leader and contains elements required for efficient RNA packaging.

The 5′-untranslated regions (5′-UTRs) of all gammaretroviruses contain a conserved “double hairpin motif” (ΨCD) that is required for genome packaging. Both hairpins (SL-C and SL-D) contain GACG tetraloops that, in isolated RNAs, are capable of forming “kissing” interactions stabilized by two intermolecular G-C base pairs. We have determined the three-dimensional structure of the double hairpin from the Moloney Murine Leukemia Virus (MoMuLV) ([ΨCD]2, 132-nucleotides, 42.8 kDaltons) using a 2H-edited NMR spectroscopy-based approach. This approach enabled the detection of 1H-1H dipolar interactions that were not observed in previous studies of isolated SL-C and SL-D hairpin RNAs using traditional 1H-1H correlated and 1H-13C-edited NMR methods. The hairpins participate in intermolecular cross-kissing interactions (SL-C to SL-D’ and SLC’ to SL-D), and stack in an end-to-end manner (SL-C to SL-D and SL-C’ to SL-D’) that gives rise to an elongated overall shape (ca. 95 Å by 45 Å by 25 Å). The global structure was confirmed by cryo-electron tomography (cryo-ET), making [ΨCD]2 simultaneously the smallest RNA to be structurally characterized to date by cryo-ET and among the largest to be determined by NMR. Our findings suggest that, in addition to promoting dimerization, [ΨCD]2 functions as a scaffold that helps initiate virus assembly by exposing a cluster of conserved UCUG elements for binding to the cognate nucleocapsid domains of assembling viral Gag proteins.

Moloney murine leukemia virus (MLV) selectively encapsidates host mY1 and mY3 RNAs. These noncoding RNA polymerase III transcripts are normally complexed with the Ro60 and La proteins, which are autoantigens associated with rheumatic disease that function in RNA biogenesis and quality control. Here, MLV replication and mY RNA packaging were analyzed using Ro60 knockout embryonic fibroblasts, which contain only ϳ3% as much mY RNA as wild-type cells. Virus spread at the same rate in wild-type and Ro knockout cells. Surprisingly, MLV virions shed by Ro60 knockout cells continued to package high levels of mY1 and mY3 (about two copies of each) like those from wild-type cells, even though mY RNAs were barely detectable within producer cells. As a result, for MLV produced in Ro60 knockout cells, encapsidation selectivity from among all cell RNAs was even higher for mY RNAs than for the viral genome. Whereas mY RNAs are largely cytoplasmic in wild-type cells, fractionation of knockout cells revealed that the residual mY RNAs were relatively abundant in nuclei, likely reflecting the fact that most mY RNAs were degraded shortly after transcription in the absence of Ro60. Together, these data suggest that these small, labile host RNAs may be recruited at a very early stage of their biogenesis and may indicate an intersection of retroviral assembly and RNA quality control pathways.

Reduced levels of survival motor neuron (SMN) protein lead to a neuromuscular disease called spinal muscular atrophy (SMA). Animal models of SMA recapitulate many aspects of the human disease, including locomotion and viability defects, but have thus far failed to uncover the causative link between a lack of SMN protein and neuromuscular dysfunction. While SMN is known to assemble small nuclear ribonucleoproteins (snRNPs) that catalyze pre-mRNA splicing, it remains unclear whether disruptions in splicing are etiologic for SMA. To investigate this issue, we carried out RNA deep-sequencing (RNA-seq) on agematched Drosophila Smn-null and wild-type larvae. Comparison of genome-wide mRNA expression profiles with publicly available data sets revealed the timing of a developmental arrest in the Smn mutants. Furthermore, genome-wide differences in splicing between wild-type and Smn animals did not correlate with changes in mRNA levels. Specifically, we found that mRNA levels of genes that contain minor introns vary more over developmental time than they do between wild-type and Smn mutants. An analysis of reads mapping to minor-class intron-exon junctions revealed only small changes in the splicing of minor introns in Smn larvae, within the normal fluctuations that occur throughout development. In contrast, Smn mutants displayed a prominent increase in levels of stress-responsive transcripts, indicating a systemic response to the developmental arrest induced by loss of SMN protein. These findings not only provide important mechanistic insight into the developmental arrest displayed by Smn mutants, but also argue against a minor-intron-dependent etiology for SMA.

Retroviruses selectively package two copies of their RNA genomes by mechanisms that have yet to be fully deciphered. Recent studies with small fragments of the Moloney murine leukaemia virus (MoMuLV) genome suggested that selection may be mediated by an RNA switch mechanism, in which conserved UCUG elements that are sequestered by base pairing in the monomeric RNA become exposed upon dimerization to allow binding to the cognate nucleocapsid (NC) domains of the viral Gag proteins. Here we show that a large fragment of the MoMuLV 5'-untranslated region (5'-UTR) that contains all residues necessary for efficient RNA packaging (Ψ WT , residues 147-623) also exhibits dimerization-dependent affinity for NC, with the native dimer ([Ψ WT ] 2 ) binding 12 ± 2 NC molecules with high affinity (K d = 17 ± 7 nM) and the monomer, stabilized by substitution of dimer-promoting loop residues by hairpin-stabilizing sequences (Ψ M ), binding 1-2 NC molecules. Identical dimer-inhibiting mutations in MoMuLV-based vectors significantly inhibit genome packaging in vivo (~100-fold decrease), whereas a large deletion of nearly 200 nucleotides just upstream of the gag start codon has minimal effects. Our findings support the proposed RNA switch mechanism, and further suggest that virus assembly may be initiated by a complex comprising as few as 12 Gag molecules bound to a dimeric packaging signal.

Survival motor neuron (SMN) functions in the assembly of spliceosomal small nuclear ribonucleoproteins (snRNPs) that catalyze pre-mRNA splicing. Here, we used disruptions in Smn and two additional snRNP biogenesis genes, Phax and Ars2, to classify RNA processing differences as snRNP-dependent or gene-specific in Drosophila. Phax and Smn mutants exhibited comparable reductions in snRNAs, and comparison of their transcriptomes uncovered shared sets of RNA processing changes. In contrast, Ars2 mutants displayed only small decreases in snRNA levels, and RNA processing changes in these mutants were generally distinct from those identified in Phax and Smn animals. Instead, RNA processing changes in Ars2 mutants support the known interaction of Ars2 protein with the cap-binding complex, as splicing changes showed a clear bias toward the first intron. Bypassing disruptions in snRNP biogenesis, direct knockdown of spliceosomal proteins caused similar changes in the splicing of snRNP-dependent events. However, these snRNP-dependent events were largely unaltered in three Smn mutants expressing missense mutations that were originally identified in human spinal muscular atrophy (SMA) patients. Hence, findings here clarify the contributions of Phax, Smn, and Ars2 to snRNP biogenesis in Drosophila, and loss-of-function mutants for these proteins reveal differences that help disentangle cause and effect in SMA model flies.

Murine leukemia virus (MLV) specifically packages both genomic RNA (FL RNA) and a subgenomic RNA, which we call SD9. SD9 RNA results from alternative splicing of FL RNA. It is reverse-transcribed, and its DNA copy, integrated into the host genome, constitutes a splice donor-associated retroelement. FL and SD9 RNAs share a common 59-UTR that includes the packaging/dimerization signal (Psi). To investigate whether the mechanism of copackaging of these two RNAs involves RNA heterodimerization, we examined the spontaneous dimerization capacity of the two RNAs as large synthetic RNAs transcribed in vitro. We showed that SD9 RNA not only formed homodimers with similar efficiency as the FL RNA, but that FL and SD9 RNAs also formed FL/SD9 heterodimers via Psi sequences. Comparison of the thermostabilities determined for these different dimeric species and competition experiments with Psi RNA fragments indicate the recruitment of similar dimer-linkage interactions within the Psi region. To validate these results, the dimeric state of the SD9 RNA was analyzed in MLV particles. RNA capture assays performed with the FL RNA as bait revealed that SD9, and not the host packageable U6 or 7SL RNAs, was associated with the FL RNA in virions. Heterodimerization of SD9 RNA with FL RNA may argue for the recent concept of a nuclear dimerization at or near the site of transcription and raises the new hypothesis of RNA dimerization during splicing. Furthermore, FL/SD9 heterodimerization may have leukemogenic consequences by influencing the pool of genomic dimers that will undergo recombinogenic template switching by reverse transcriptase.