Transcription Start Site Usage Research Articles

Transcription start site scanning requires the fungi-specific hydrophobic loop of Tfb3.

RNA polymerase II (pol II) initiates transcription from transcription start sites (TSSs) located ∼30-35 bp downstream of the TATA box in metazoans, whereas in the yeast Saccharomyces cerevisiae, pol II scans further downstream TSSs located ∼40-120 bp downstream of the TATA box. Previously, we found that removal of the kinase module TFIIK (Kin28-Ccl1-Tfb3) from TFIIH shifts the TSS in a yeast in vitro system upstream to the location observed in metazoans and that addition of recombinant Tfb3 back to TFIIH-ΔTFIIK restores the downstream TSS usage. Here, we report that this biochemical activity of yeast TFIIK in TSS scanning is attributable to the Tfb3 RING domain at the interface with pol II in the pre-initiation complex (PIC): especially, swapping Tfb3 Pro51-a residue conserved among all fungi-with Ala or Ser as in MAT1, the metazoan homolog of Tfb3, confers an upstream TSS shift in vitro in a similar manner to the removal of TFIIK. Yeast genetic analysis suggests that both Pro51 and Arg64 of Tfb3 are required to maintain the stability of the Tfb3-pol II interface in the PIC. Cryo-electron microscopy analysis of a yeast PIC lacking TFIIK reveals considerable variability in theorientation of TFIIH, which impairs TSS scanning after promoter opening.

Role of RNA structural plasticity in modulating HIV-1 genome packaging and translation

HIV-1 transcript function is controlled in part by twinned transcriptional start site usage, where 5' capped RNAs beginning with a single guanosine (1G) are preferentially packaged into progeny virions as genomic RNA (gRNA) whereas those beginning with three sequential guanosines (3G) are retained in cells as mRNAs. In 3G transcripts, one of the additional guanosines base pairs with a cytosine located within a conserved 5' polyA element, resulting in formation of an extended 5' polyA structure as opposed to the hairpin structure formed in 1G RNAs. To understand how this remodeling influences overall transcript function, we applied in vitro biophysical studies with in-cell genome packaging and competitive translation assays to native and 5' polyA mutant transcripts generated with promoters that differentially produce 1G or 3G RNAs. We identified mutations that stabilize the 5' polyA hairpin structure in 3G RNAs, which promote RNA dimerization and Gag binding without sequestering the 5' cap. None of these 3G transcripts were competitively packaged, confirming that cap exposure is a dominant negative determinant of viral genome packaging. For all RNAs examined, conformations that favored 5' cap exposure were both poorly packaged and more efficiently translated than those that favored 5' cap sequestration. We propose that structural plasticity of 5' polyA and other conserved RNA elements place the 5' leader on a thermodynamic tipping point for low-energetic (~3 kcal/mol) control of global transcript structure and function.

Characterizing a complex CT-rich haplotype in intron 4 of SNCA using large-scale targeted amplicon long-read sequencing

Parkinson’s disease (PD) is a common neurodegenerative disorder with a significant risk proportion driven by genetics. While much progress has been made, most of the heritability remains unknown. This is in-part because previous genetic studies have focused on the contribution of single nucleotide variants. More complex forms of variation, such as structural variants and tandem repeats, are already associated with several synucleinopathies. However, because more sophisticated sequencing methods are usually required to detect these regions, little is understood regarding their contribution to PD. One example is a polymorphic CT-rich region in intron 4 of the SNCA gene. This haplotype has been suggested to be associated with risk of Lewy Body (LB) pathology in Alzheimer’s Disease and SNCA gene expression, but is yet to be investigated in PD. Here, we attempt to resolve this CT-rich haplotype and investigate its role in PD. We performed targeted PacBio HiFi sequencing of the region in 1375 PD cases and 959 controls. We replicate the previously reported associations and a novel association between two PD risk SNVs (rs356182 and rs5019538) and haplotype 4, the largest haplotype. Through quantitative trait locus analyzes we identify a significant haplotype 4 association with alternative CAGE transcriptional start site usage, not leading to significant differential SNCA gene expression in post-mortem frontal cortex brain tissue. Therefore, disease association in this locus might not be biologically driven by this CT-rich repeat region. Our data demonstrates the complexity of this SNCA region and highlights that further follow up functional studies are warranted.

Alternative TSS use is widespread in Cryptococcus fungi in response to environmental cues and regulated genome-wide by the transcription factor Tur1.

Alternative transcription start site (TSS) usage regulation has been identified as a major means of gene expression regulation in metazoans. However, in fungi, its impact remains elusive as its study has thus far been restricted to model yeasts. Here, we first re-analyzed TSS-seq data to define genuine TSS clusters in 2 species of pathogenic Cryptococcus. We identified 2 types of TSS clusters associated with specific DNA sequence motifs. Our analysis also revealed that alternative TSS usage regulation in response to environmental cues is widespread in Cryptococcus, altering gene expression and protein targeting. Importantly, we performed a forward genetic screen to identify a unique transcription factor (TF) named Tur1, which regulates alternative TSS (altTSS) usage genome-wide when cells switch from exponential phase to stationary phase. ChiP-Seq and DamID-Seq analyses suggest that at some loci, the role of Tur1 might be direct. Tur1 has been previously shown to be essential for virulence in C. neoformans. We demonstrated here that a tur1Δ mutant strain is more sensitive to superoxide stress and phagocytosed more efficiently by macrophages than the wild-type (WT) strain.

Pan-cancer transcriptome analysis reveals widespread regulation through alternative tandem transcription initiation.

Abnormal transcription initiation from alternative first exon has been reported to promote tumorigenesis. However, the prevalence and impact of gene expression regulation mediated by alternative tandem transcription initiation were mostly unknown in cancer. Here, we developed a robust computational method to analyze alternative tandem transcription start site (TSS) usage from standard RNA sequencing data. Applying this method to pan-cancer RNA sequencing datasets, we observed widespread dysregulation of tandem TSS usage in tumors, many of which were independent of changes in overall expression level or alternative first exon usage. We showed that the dynamics of tandem TSS usage was associated with epigenomic modulation. We found that significant 5' untranslated region shortening of gene TIMM13 contributed to increased protein production, and up-regulation of TIMM13 by CRISPR-mediated transcriptional activation promoted proliferation and migration of lung cancer cells. Our findings suggest that dysregulated tandem TSS usage represents an addtional layer of cancer-associated transcriptome alterations.

Deciphering the cis-regulatory landscape of natural yeast Transcript Leaders.

Protein synthesis is a vital process that is highly regulated at the initiation step of translation. Eukaryotic 5' transcript leaders (TLs) contain a variety of cis-regulatory features that influence translation and mRNA stability. However, the relative influences of these features in natural TLs are poorly characterized. To address this, we used massively parallel reporter assays (MPRAs) to quantify RNA levels, ribosome loading, and protein levels from 11,027 natural yeast TLs in vivo and systematically compared the relative impacts of their sequence features on gene expression. We found that yeast TLs influence gene expression over two orders of magnitude. While a leaky scanning model using Kozak contexts and uAUGs explained half of the variance in expression across transcript leaders, the addition of other features explained ~70% of gene expression variation. Our analyses detected key cis-acting sequence features, quantified their effects in vivo, and compared their roles to motifs reported from an in vitro study of ribosome recruitment. In addition, our work quantitated the effects of alternative transcription start site usage on gene expression in yeast. Thus, our study provides new quantitative insights into the roles of TL cis-acting sequences in regulating gene expression.

Uncovering the dynamics and consequences of RNA isoform changes during neuronal differentiation

Static gene expression programs have been extensively characterized in stem cells and mature human cells. However, the dynamics of RNA isoform changes upon cell-state-transitions during cell differentiation, the determinants and functional consequences have largely remained unclear. Here, we established an improved model for human neurogenesis in vitro that is amenable for systems-wide analyses of gene expression. Our multi-omics analysis reveals that the pronounced alterations in cell morphology correlate strongly with widespread changes in RNA isoform expression. Our approach identifies thousands of new RNA isoforms that are expressed at distinct differentiation stages. RNA isoforms mainly arise from exon skipping and the alternative usage of transcription start and polyadenylation sites during human neurogenesis. The transcript isoform changes can remodel the identity and functions of protein isoforms. Finally, our study identifies a set of RNA binding proteins as a potential determinant of differentiation stage-specific global isoform changes. This work supports the view of regulated isoform changes that underlie state-transitions during neurogenesis.

A Bayesian Change Point Model for Dynamic Alternative Transcription Start Site Usage During Cellular Differentiation.

ABSTRACT An alternative transcription start site (ATSS) is a major driving force for increasing the complexity of transcripts in human tissues. As a transcriptional regulatory mechanism, ATSS has biological significance. Many studies have confirmed that ATSS plays an important role in diseases and cell development and differentiation. However, exploration of its dynamic mechanisms remains insufficient. Identifying ATSS change points during cell differentiation is critical for elucidating potential dynamic mechanisms. For relative ATSS usage as percentage data, the existing methods lack sensitivity to detect the change point for ATSS longitudinal data. In addition, some methods have strict requirements for data distribution and cannot be applied to deal with this problem. In this study, the Bayesian change point detection model was first constructed using reparameterization techniques for two parameters of a beta distribution for the percentage data type, and the posterior distributions of parameters and change points were obtained using Markov Chain Monte Carlo (MCMC) sampling. With comprehensive simulation studies, the performance of the Bayesian change point detection model is found to be consistently powerful and robust across most scenarios with different sample sizes and beta distributions. Second, differential ATSS events in the real data, whose change points were identified using our method, were clustered according to their change points. Last, for each change point, pathway and transcription factor motif analyses were performed on its differential ATSS events. The results of our analyses demonstrated the effectiveness of the Bayesian change point detection model and provided biological insights into cell differentiation.

A single-cell genomic strategy for alternative transcript start sites identification.

Alternative transcription start sites (TSSs) usage plays a critical role in gene transcription regulation in mammals. However, precisely identifying alternative TSSs remains challenging at the genome-wide level. We report a single-cell genomic technology for alternative TSSs annotation and cell heterogeneity detection. In the method, we utilize Fluidigm C1 system to capture individual cells of interest, SMARTer cDNA synthesis kit to recover full-length cDNAs, then dual priming oligonucleotide system to specifically enrich TSSs for genomic analysis. We apply this method to a genome-wide study of alternative TSSs identification in two different IFN-β stimulated mouse embryonic fibroblasts (MEFs). The data clearly discriminate two IFN-β stimulated MEFs. Moreover, our results indicate 81% expressed genes in these two cell types containing multiple TSSs, which is much higher than previous predictions based on Cap-Analysis Gene Expression (CAGE) (58%) or empirical determination (54%) in various cell types. This indicates that alternative TSSs are more pervasive than expected and implies our strategy could position them at an unprecedented sensitivity. It would be helpful for elucidating their biological insights in future.

TE-TSS: an integrated data resource of human and mouse transposable element (TE)-derived transcription start site (TSS).

Transposable elements (TEs) are abundant in the genome and serve as crucial regulatory elements. Some TEs function as epigenetically regulated promoters, and these TE-derived transcription start sites (TSSs) play a crucial role in regulating genes associated with specific functions, such as cancer and embryogenesis. However, the lack of an accessible database that systematically gathers TE-derived TSS data is a current research gap. To address this, we established TE-TSS, an integrated data resource of human and mouse TE-derived TSSs (http://xozhanglab.com/TETSS). TE-TSS has compiled 2681 RNA sequencing datasets, spanning various tissues, cell linesand developmental stages. From these, we identified 5768 human TE-derived TSSs and 2797 mouse TE-derived TSSs, with 47% and 38% being experimentally validated, respectively. TE-TSS enables comprehensive exploration of TSS usage in diverse samples, providing insights into tissue-specific gene expression patterns and transcriptional regulatory elements. Furthermore, TE-TSS compares TE-derived TSS regions across 15 mammalian species, enhancing our understanding of their evolutionary and functional aspects. The establishment of TE-TSS facilitates further investigations into the roles of TEs in shaping the transcriptomic landscape and offers valuable resources for comprehending their involvement in diverse biological processes.

CamoTSS: analysis of alternative transcription start sites for cellular phenotypes and regulatory patterns from 5' scRNA-seq data

Five-prime single-cell RNA-seq (scRNA-seq) has been widely employed to profile cellular transcriptomes, however, its power of analysing transcription start sites (TSS) has not been fully utilised. Here, we present a computational method suite, CamoTSS, to precisely identify TSS and quantify its expression by leveraging the cDNA on read 1, which enables effective detection of alternative TSS usage. With various experimental data sets, we have demonstrated that CamoTSS can accurately identify TSS and the detected alternative TSS usages showed strong specificity in different biological processes, including cell types across human organs, the development of human thymus, and cancer conditions. As evidenced in nasopharyngeal cancer, alternative TSS usage can also reveal regulatory patterns including systematic TSS dysregulations.

Transcription start site heterogeneity and its role in RNA fate determination distinguish HIV-1 from other retroviruses and are mediated by core promoter elements.

HIV-1 uses heterogeneous transcription start sites (TSSs) to generate two RNA 5´ isoforms that adopt radically different structures and perform distinct replication functions. Although these RNAs differ in length by only two bases, exclusively, the shorter RNA is encapsidated while the longer RNA is excluded from virions and provides intracellular functions. The current study examined TSS usage and packaging selectivity for a broad range of retroviruses and found that heterogeneous TSS usage was a conserved feature of all tested HIV-1 strains, but all other retroviruses examined displayed unique TSSs. Phylogenetic comparisons and chimeric viruses' properties provided evidence that this mechanism of RNA fate determination was an innovation of the HIV-1 lineage, with determinants mapping to core promoter elements. Fine-tuning differences between HIV-1 and HIV-2, which uses a unique TSS, implicated purine residue positioning plus a specific TSS-adjacent dinucleotide in specifying multiplicity of TSS usage. Based on these findings, HIV-1 expression constructs were generated that differed from the parental strain by only two point mutations yet each expressed only one of HIV-1's two RNAs. Replication defects of the variant with only the presumptive founder TSS were less severe than those for the virus with only the secondary start site. IMPORTANCE Retroviruses use RNA both to encode their proteins and to serve in place of DNA as their genomes. A recent surprising discovery was that the genomic RNAs and messenger RNAs of HIV-1 are not identical but instead differ subtly on one of their ends. These differences enable the functional separation of HIV-1 RNAs into genome and messenger roles. In this report, we examined a broad collection of HIV-1-related viruses and discovered that each produced only one end class of RNA, and thus must differ from HIV-1 in how they specify RNA fates. By comparing regulatory signals, we generated virus variants that pinpointed the determinants of HIV-1 RNA fates, as well as HIV-1 variants that produced only one or the other functional class of RNA. Competition and replication assays confirmed that HIV-1 has evolved to rely on the coordinated actions of both its RNA forms.

Multi-landmark alignment of genomic signals reveals conserved expression patterns across transcription start sites

The prevalent one-dimensional alignment of genomic signals to a reference landmark is a cornerstone of current methods to study transcription and its DNA-dependent processes but it is prone to mask potential relations among multiple DNA elements. We developed a systematic approach to align genomic signals to multiple locations simultaneously by expanding the dimensionality of the genomic-coordinate space. We analyzed transcription in human and uncovered a complex dependence on the relative position of neighboring transcription start sites (TSSs) that is consistently conserved among cell types. The dependence ranges from enhancement to suppression of transcription depending on the relative distances to the TSSs, their intragenic position, and the transcriptional activity of the gene. Our results reveal a conserved hierarchy of alternative TSS usage within a previously unrecognized level of genomic organization and provide a general methodology to analyze complex functional relationships among multiple types of DNA elements.

The single-cell landscape of alternative transcription start sites of diabetic retina

More than half of mammalian protein-coding genes have multiple transcription start sites. Alternative transcription start site (TSS) modulate mRNA stability, localization, and translation efficiency on post-transcription level, and even generate novel protein isoforms. However, differential TSS usage among cell types in healthy and diabetic retina remains poorly characterized. In this study, by using 5′-tag-based single-cell RNA sequencing, we identified cell type-specific alternative TSS events and key transcription factors for each of retinal cell types. We observed that lengthening of 5′- UTRs in retinal cell types are enriched for multiple RNA binding protein binding sites, including splicing regulators Rbfox1/2/3 and Nova1. Furthermore, by comparing TSS expression between healthy and diabetic retina, we identified elevated apoptosis signal in Müller glia and microglia, which can be served as a putative early sign of diabetic retinopathy. By measuring 5′UTR isoforms in retinal single-cell dataset, our work provides a comprehensive panorama of alternative TSS and its potential consequence related to post-transcriptional regulation. We anticipate our assay can not only provide insights into cellular heterogeneity driven by transcriptional initiation, but also open up the perspectives for identification of novel diagnostic indexes for diabetic retinopathy.

PO-01-145 DYNAMIC UTR COMPOSITION IMPACTS RNA BINDING PROTEIN COMPLEXING TO CONTROL TRANSLATION AND LIMIT CARDIAC GAP JUNCTION FORMATION DURING STRESS

Translational control has emerged as a critical yet dynamic process, representing one of the most rapid means by which the cardiomyocyte can alter its proteome. Several key cardiac mRNAs, including connexin43 (Cx43, gene name GJA1) are understood to undergo alternative translation initiation with resulting products (ie, GJA1-20k) impacting cardiac function. We previously found alterations to GJA1 mRNA untranslated region (UTR) length occur in aged and stressed heart tissue, suppressing internal translation of GJA1-20k, and limiting Cx43 gap junction formation. Having therefore identified dynamic mRNA cis element-based regulation of Cx43/GJA1-20k translation, we next asked what trans-acting factors are at play to alter GJA1 mRNA translation during stress. The IMP family of RNA binding proteins (RBPs) regulate alternative and canonical translation initiation of several genes, which led us to investigate how they may impact GJA1 mRNA. Using GJA1 as a model RNA, we test the hypothesis that alternative transcription start site usage controls inclusion of cis-acting elements in the mRNA 5’UTR affecting RBP binding and regulation of translation. Pathological hypertrophy was induced by isoproterenol deliverey to adult mice via implanted minipumps. Mouse models are complemented with matured human IPSC-derived cardiomyocytes, primary mouse ventricular cardiomyocytes, exposed to chronic hypoxia or TGF-β in vitro. Single molecule fluorescence in situ hybridization (smFISH) coupled with super-resolution microscopy localize GJA1 mRNA and RBPs. Hypertrophic hearts show reduced GJA1-20k and IMP1 expression. Using smFISH to label GJA1 mRNA, we find IMP1 complexes with GJA1 mRNA predominantly in comparison to IMP2 and 3, and that this complexing is reduced during stress, concomitant with loss of GJA1-20k internal translation. Biochemical assays confirm GJA1 mRNA/IMP1 binding by RNA-IP and synthetic reduction of IMP1 expression by RNAi or CRISPR/Cas9 alters GJA1 mRNA localization, reduces internal translation of GJA1-20k, and limits gap junction formation. We find comparable results during hypoxia in primary mouse cardiomyocytes and human induced pluripotent stem cell derived cardiomyocytes. Dynamic GJA1-5’UTR composition therefore represents a powerful mechanism connecting alterations in transcription to gene-specific translational regulation by RBPs such as IMP1, disruption of intercellular communication during stress, and arrhythmogenesis.

Implications of differential transcription start site selection on chronic myeloid leukemia and prostate cancer cell protein expression.

The relevance of minor transcription start sites in broad promoters is not well understood. We have studied AGAP2 expression in prostate cancer and chronic myeloid leukemia (CML), showing transcription is initiated from alternative transcription start sites (TSSs) within a single TSS cluster, producing cancer-type-specific AGAP2 mRNAs with small differences in their 5' UTR length. Interestingly, in the CML cell lines where the 5' UTR is longer, AGAP2 protein levels are lower. We demonstrate that the selection of an upstream TSS involved the formation of a G quadruplex in the 5' UTR, decreasing polysome formation. After developing a bioinformatics pipeline to query data from the FANTOM project and the NCl-60 human tumor cell lines screen, we found HK1 expression can also be regulated by the same mechanism. Overall, we present compelling data supporting TSS selection within a TSS cluster play a role in protein expression and should not be ignored.

Promoter sequence and architecture determine expression variability and confer robustness to genetic variants.

Genetic and environmental exposures cause variability in gene expression. Although most genes are affected in a population, their effect sizes vary greatly, indicating the existence of regulatory mechanisms that could amplify or attenuate expression variability. Here, we investigate the relationship between the sequence and transcription start site architectures of promoters and their expression variability across human individuals. We find that expression variability can be largely explained by a promoter's DNA sequence and its binding sites for specific transcription factors. We show that promoter expression variability reflects the biological process of a gene, demonstrating a selective trade-off between stability for metabolic genes and plasticity for responsive genes and those involved in signaling. Promoters with a rigid transcription start site architecture are more prone to have variable expression and to be associated with genetic variants with large effect sizes, while a flexible usage of transcription start sites within a promoter attenuates expression variability and limits genotypic effects. Our work provides insights into the variable nature of responsive genes and reveals a novel mechanism for supplying transcriptional and mutational robustness to essential genes through multiple transcription start site regions within a promoter.

Alternative Transcription Start Site Usage and Functional Implications in Pathogenic Fungi.

Pathogenic fungi require delicate gene regulation mechanisms to adapt to diverse living environments and escape host immune systems. Recent advances in sequencing technology have exposed the complexity of the fungal genome, thus allowing the gradual disentanglement of multiple layers of gene expression control. Alternative transcription start site (aTSS) usage, previously reported to be prominent in mammals and to play important roles in physiopathology, is also present in fungi to fine-tune gene expression. Depending on the alteration in their sequences, RNA isoforms arising from aTSSs acquire different characteristics that significantly alter their stability and translational capacity as well as the properties and biologic functions of the resulting proteins. Disrupted control of aTSS usage has been reported to severely impair growth, virulence, and the infectious capacity of pathogenic fungi. Here, we discuss principle concepts, mechanisms, and the functional implication of aTSS usage in fungi.

The TRIPLE PHD FINGERS proteins are required for SWI/SNF complex-mediated +1 nucleosome positioning and transcription start site determination in Arabidopsis

Eukaryotes have evolved multiple ATP-dependent chromatin remodelers to shape the nucleosome landscape. We recently uncovered an evolutionarily conserved SWItch/Sucrose Non-Fermentable (SWI/SNF) chromatin remodeler complex in plants reminiscent of the mammalian BAF subclass, which specifically incorporates the MINUSCULE (MINU) catalytic subunits and the TRIPLE PHD FINGERS (TPF) signature subunits. Here we report experimental evidence that establishes the functional relevance of TPF proteins for the complex activity. Our results show that depletion of TPF triggers similar pleiotropic phenotypes and molecular defects to those found in minu mutants. Moreover, we report the genomic location of MINU2 and TPF proteins as representative members of this SWI/SNF complex and their impact on nucleosome positioning and transcription. These analyses unravel the binding of the complex to thousands of genes where it modulates the position of the +1 nucleosome. These targets tend to produce 5′-shifted transcripts in the tpf and minu mutants pointing to the participation of the complex in alternative transcription start site usage. Interestingly, there is a remarkable correlation between +1 nucleosome shift and 5′ transcript length change suggesting their functional connection. In summary, this study unravels the function of a plant SWI/SNF complex involved in +1 nucleosome positioning and transcription start site determination.

Transcription Start Site Heterogeneity and Preferential Packaging of Specific Full-Length RNA Species Are Conserved Features of Primate Lentiviruses.

HIV-1 must package its RNA genome to generate infectious viruses. Recent studies have revealed that during genome packaging, HIV-1 not only excludes cellular mRNAs, but also distinguishes among full-length viral RNAs. Using NL4-3 and MAL molecular clones, multiple transcription start sites (TSS) were identified, which generate full-length RNAs that differ by only a few nucleotides at the 5' end. However, HIV-1 selectively packages RNAs containing one guanosine (1G RNA) over RNAs with three guanosines (3G RNA) at the 5' end. Thus, the 5' context of HIV-1 full-length RNA can affect its function. To determine whether the regulation of genome packaging by TSS usage is unique to NL4-3 and MAL, we examined 15 primate lentiviruses including transmitted founder viruses of HIV-1, HIV-2, and several simian immunodeficiency viruses (SIVs). We found that all 15 viruses used multiple TSS to some extent. However, the level of TSS heterogeneity in infected cells varied greatly, even among closely related viruses belonging to the same subtype. Most viruses also exhibited selective packaging of specific full-length viral RNA species into particles. These findings demonstrate that TSS heterogeneity and selective packaging of certain full-length viral RNA species are conserved features of primate lentiviruses. In addition, an SIV strain closely related to the progenitor virus that gave rise to HIV-1 group M, the pandemic pathogen, exhibited TSS usage similar to some HIV-1 strains and preferentially packaged 1G RNA. These findings indicate that multiple TSS usage and selective packaging of a particular unspliced RNA species predate the emergence of HIV-1. IMPORTANCE Unspliced HIV-1 RNA serves two important roles during viral replication: as the virion genome and as the template for translation of Gag/Gag-Pol. Previous studies of two HIV-1 molecular clones have concluded that the TSS usage affects unspliced HIV-1 RNA structures and functions. To investigate the evolutionary origin of this replication strategy, we determined TSS of HIV-1 RNA in infected cells and virions for 15 primate lentiviruses. All HIV-1 isolates examined, including several transmitted founder viruses, utilized multiple TSS and selected a particular RNA species for packaging. Furthermore, these features were observed in SIVs related to the progenitors of HIV-1, suggesting that these characteristics originated from the ancestral viruses. HIV-2, SIVs related to HIV-2, and other SIVs also exhibited multiple TSS and preferential packaging of specific unspliced RNA species, demonstrating that this replication strategy is broadly conserved across primate lentiviruses.