Late RNA Research Articles

Viroids: "living fossils" of primordial RNAs?

The discovery of the viroid in 1971, which initiated the third major expansion of the biosphere towards smaller living entities—after discovery of the “subvisual” microorganisms in 1675 and that of the “submicroscopic” viruses in 1892—has been officially endorsed by the International Committee on Virus Taxonomy as a new order called subviral agents.In 1989, I proposed that, based on their respective molecular properties, viroids are more plausible “living fossils” of the hypothetical RNA World (widely assumed to have existed prior to the evolution of DNA or proteins) than are intron-derived RNAs, which were, at that time, suggested as putative survivors. There were few citations of my proposal—and virtually none of viroids—beyond plant virology unil 1994, when Cheles-Flores critically examined the hypothesis and pointed out a serious difficulty, as well as a process by which this difficulty could be overcome. In 2013, when investigations by Koonin and Dolja revealed that of extant RNAs, viroids “strikingly” display some of the molecular properties posited for the earliest evolving, selfish RNAs (primordial RNAs), but, because extant organisms, aside from higher plants, appear not to harbor viroids, they cannot be regarded as primordial fossils, but appear to have evolved post LUCA (the Last Universal Common Ancestor). Here, I review whether some evidence nevertheless is compatible with the original postulate of the 1989 hypothesis. My analysis reveals no unequivocal evidence for an ancient origin of viroids, but suggests, alternatively, that viroids may have evolved de novo more recently, probably by novel processes similar to those suggested by each reviewer.These results are important, because they help illuminate a little understood period of abiogenesis—after the abiotic synthesis of life’s chemical building blocks, which is, in principle, understood, and before the evolution of DNA and proteins in the late RNA World.

A cluster of 3' coterminal transcripts from US12-US17 locus of human cytomegalovirus.

Among all the human cytomegalovirus (HCMV) gene families, US12 family is relatively undefined in their transcriptional profile and biological functions. In this study, the transcription pattern and characteristics of HCMV US12-US17 gene region were studied extensively. Twenty-three clones harboring US12 cDNA sequence were screened out from a late cDNA library of an HCMV clinical isolate, Han. Using a set of US12-US17 gene-specific probes, six transcripts from US12-US17 locus were detected by northern blot at late kinetics of the clinical isolate. One additional transcript was found in late RNA of HCMV strain AD169. No evidence showing these transcripts contain introns by reverse transcription PCR. 3' and 5' termini of these transcripts were confirmed by Rapid Amplification of cDNA Ends. A novel protein-coding region was predicted in the shorter US14 transcript with an alternative in-frame 5' translation initiation site compared to that of the previously predicted US14 ORF. Our findings demonstrate the existence of a cluster of 3' coterminal unspliced transcripts with distinct 5' transcriptional initiation sites originated from US12-US17 gene region in the late infection phase of an HCMV clinical strain.

Human Exportin-1 is a Target for Combined Therapy of HIV and AIDS Related Lymphoma

Infection with HIV ultimately leads to advanced immunodeficiency resulting in an increased incidence of cancer. For example primary effusion lymphoma (PEL) is an aggressive non-Hodgkin lymphoma with very poor prognosis that typically affects HIV infected individuals in advanced stages of immunodeficiency. Here we report on the dual anti-HIV and anti-PEL effect of targeting a single process common in both diseases. Inhibition of the exportin-1 (XPO1) mediated nuclear transport by clinical stage orally bioavailable small molecule inhibitors (SINE) prevented the nuclear export of the late intron-containing HIV RNA species and consequently potently suppressed viral replication. In contrast, in CRISPR-Cas9 genome edited cells expressing mutant C528S XPO1, viral replication was unaffected upon treatment, clearly demonstrating the anti-XPO1 mechanism of action. At the same time, SINE caused the nuclear accumulation of p53 tumor suppressor protein as well as inhibition of NF-κB activity in PEL cells resulting in cell cycle arrest and effective apoptosis induction. In vivo, oral administration arrested PEL tumor growth in engrafted mice. Our findings provide strong rationale for inhibiting XPO1 as an innovative strategy for the combined anti-retroviral and anti-neoplastic treatment of HIV and PEL and offer perspectives for the treatment of other AIDS-associated cancers and potentially other virus-related malignancies.

Newly identified transcripts of UL4 and UL5 genes of human cytomegalovirus.

Human cytomegalovirus (HCMV) UL4 and UL5 genes are two members of the RL11 gene family. In an earlier study, three UL4 transcripts of about 1.7, 1.5 and 1.4 kb were found in early and late classes after infection by the Towne strain by nuclease protection and primer extension analyses. In the present study, two UL4 transcripts (1.5 and 1.7 kb) were found by cDNA library screening, Northern blot, 3' and 5' RACE analyses to appear initially in the immediate early phase and one UL4 transcript (1.4 kb) in the late phase in a low-passage clinical isolate. Furthermore, two novel low-abundance UL5 transcripts with the same 3' terminus as the identified UL4 transcripts in the UL4-UL5 gene region were found in late class RNAs.

WBSCR22/Merm1 is required for late nuclear pre-ribosomal RNA processing and mediates N7-methylation of G1639 in human 18S rRNA.

Ribosomal (r)RNAs are extensively modified during ribosome synthesis and their modification is required for the fidelity and efficiency of translation. Besides numerous small nucleolar RNA-guided 2′-O methylations and pseudouridinylations, a number of individual RNA methyltransferases are involved in rRNA modification. WBSCR22/Merm1, which is affected in Williams–Beuren syndrome and has been implicated in tumorigenesis and metastasis formation, was recently shown to be involved in ribosome synthesis, but its molecular functions have remained elusive. Here we show that depletion of WBSCR22 leads to nuclear accumulation of 3′-extended 18SE pre-rRNA intermediates resulting in impaired 18S rRNA maturation. We map the 3′ ends of the 18SE pre-rRNA intermediates accumulating after depletion of WBSCR22 and in control cells using 3′-RACE and deep sequencing. Furthermore, we demonstrate that WBSCR22 is required for N7-methylation of G1639 in human 18S rRNA in vivo. Interestingly, the catalytic activity of WBSCR22 is not required for 18S pre-rRNA processing, suggesting that the key role of WBSCR22 in 40S subunit biogenesis is independent of its function as an RNA methyltransferase.

Late HDV RNA relapse after peginterferon alpha-based therapy of chronic hepatitis delta

Interferon alpha is the only treatment option for hepatitis delta virus (HDV). Trials investigating the efficacy of pegylated interferon alpha (PEG-IFNa) showed HDV RNA negativity rates of 25-30% 24 weeks after therapy. However, the clinical and virological long-term outcome of HDV-infected patients treated with PEG-IFNa is unknown. We performed a retrospective-prospective follow-up of 77 patients treated for 48 weeks with either PEG-alfa-2a and adefovir (ADV) or either drug alone in the Hep-Net-International-Delta-Hepatitis-Intervention-Study 1 (HIDIT-1) trial. Long-term follow-up data were available for 58 out of 77 patients (75%) with a median time of follow-up of 4.5 (0.5-5.5) years and a median 3 visits per patient. Patients treated with ADV alone received retreatment with PEG-IFNa (48% versus 19%; P = 0.02) more often. Hepatitis B virus surface antigen (HBsAg) became negative in six PEG-IFNa-treated patients until the end of long-term follow-up (10%). Sixteen patients tested HDV RNA-negative 6 months after PEG-IFNa treatment who were entered in the long-term follow-up study. Out of these, nine individuals tested HDV RNA-positive at least once during further long-term follow-up, with seven patients being HDV RNA-positive at the most recent visit. Clinical endpoints (liver-related death, liver transplantation, hepatic decompensation, hepatocellular carcinoma) were observed in three PEG-IFNa-treated (8%) and three ADV-treated (14%) patients during posttreatment long-term follow-up with an overall annual event rate of 2.5% (4.9% in cirrhosis). Sequencing confirmed the reappearance of pretreatment virus strains in all cases. Late HDV RNA relapses may occur after PEG-IFNa therapy of hepatitis delta and thus the term sustained virological response should be avoided in HDV infection. The annual posttreatment rate of clinical events in hepatitis delta patients eligible for PEG-IFNa therapy is about 2.5% and 4.9% in patients with cirrhosis.

Localisation of RNAs into the Germ Plasm of Vitellogenic Xenopus Oocytes

We have studied the localisation of mRNAs in full-grown Xenopus laevis oocytes by injecting fluorescent RNAs, followed by confocal microscopy of the oocyte cortex. Concentrating on RNA encoding the Xenopus Nanos homologue, nanos1 (formerly Xcat2), we find that it consistently localised into aggregated germ plasm ribonucleoprotein (RNP) particles, independently of cytoskeletal integrity. This implies that a diffusion/entrapment-mediated mechanism is active, as previously reported for previtellogenic oocytes. Sometimes this was accompanied by localisation into scattered particles of the “late”, Vg1/VegT pathway; occasionally only late pathway localisation was seen. The Xpat RNA behaved in an identical fashion and for neither RNA was the localisation changed by any culture conditions tested. The identity of the labelled RNP aggregates as definitive germ plasm was confirmed by their inclusion of abundant mitochondria and co-localisation with the germ plasm protein Hermes. Further, the nanos1/Hermes RNP particles are interspersed with those containing the germ plasm protein Xpat. These aggregates may be followed into the germ plasm of unfertilized eggs, but with a notable reduction in its quantity, both in terms of injected molecules and endogenous structures. Our results conflict with previous reports that there is no RNA localisation in large oocytes, and that during mid-oogenesis even germ plasm RNAs localise exclusively by the late pathway. We find that in mid oogenesis nanos1 RNA also localises to germ plasm but also by the late pathway. Late pathway RNAs, Vg1 and VegT, also may localise into germ plasm. Our results support the view that mechanistically the two modes of localisation are extremely similar, and that in an injection experiment RNAs might utilise either pathway, the distinction in fates being very subtle and subject to variation. We discuss these results in relation to their biological significance and the results of others.

Non-coding RNAs in Crop Genetic Modification: Considerations and Predictable Environmental Risk Assessments (ERA)

Of late non-coding RNAs (ncRNAs)-mediated gene silencing is an influential tool deliberately deployed to negatively regulate the expression of targeted genes. In addition to the widely employed small interfering RNA (siRNA)-mediated gene silencing approach, other variants like artificial miRNA (amiRNA), miRNA mimics, and artificial transacting siRNAs (tasiRNAs) are being explored and successfully deployed in developing non-coding RNA-based genetically modified plants. The ncRNA-based gene manipulations are typified with mobile nature of silencing signals, interference from viral genome-derived suppressor proteins, and an obligation for meticulous computational analysis to prevaricate any inadvertent effects. In a broad sense, risk assessment inquiries for genetically modified plants based on the expression of ncRNAs are competently addressed by the environmental risk assessment (ERA) models, currently in vogue, designed for the first generation transgenic plants which are based on the expression of heterologous proteins. Nevertheless, transgenic plants functioning on the foundation of ncRNAs warrant due attention with respect to their unique attributes like off-target or non-target gene silencing effects, small RNAs (sRNAs) persistence, food and feed safety assessments, problems in detection and tracking of sRNAs in food, impact of ncRNAs in plant protection measures, effect of mutations etc. The role of recent developments in sequencing techniques like next generation sequencing (NGS) and the ERA paradigm of the different countries in vogue are also discussed in the context of ncRNA-based gene manipulations.

Overlapping transcription structure of human cytomegalovirus UL140 and UL141 genes

Transcription of human cytomegalovirus UL/b' region has been studied extensively for some genes. In this study, transcripts of the UL140 and UL141, two of the UL/b' genes, were identified in late RNAs of three HCMV isolates using Northern blot hybridization, cDNA library screening and RACE-PCR. At least three transcripts with length of 2800, 2400 and 1700 nt, as well as a group of transcripts of about 1000-1300 nt, were found in this gene region with an accordant 3' ends. Among the transcripts, two initiated upstream of the start code of the UL140 gene and contained the UL140 and UL141 open reading frame (ORF), one initiated in the middle of the UL140 gene, and could encode short ORFs upstream of the UL141 ORF. A group of transcripts initiated upstream or downstream of the start code of the UL141 gene, and could encode 'nested' ORFs, including the UL141 ORF. These 'nested' ORFs possess different initiation sites but the same termination site as that of the UL141 ORF.

Pervasive Initiation and 3′-End Formation of Poxvirus Postreplicative RNAs

Poxviruses are large DNA viruses that replicate within the cytoplasm and encode a complete transcription system, including a multisubunit RNA polymerase, stage-specific transcription factors, capping and methylating enzymes, and a poly(A) polymerase. Expression of the more than 200 open reading frames by vaccinia virus, the prototype poxvirus, is temporally regulated: early mRNAs are synthesized immediately after infection, whereas intermediate and late mRNAs are synthesized following genome replication. The postreplicative transcripts are heterogeneous in length and overlap the entire genome, which pose obstacles for high resolution mapping. We used tag-based methods in conjunction with high throughput cDNA sequencing to determine the precise 5'-capped and 3'-polyadenylated ends of postreplicative RNAs. Polymerase slippage during initiation of intermediate and late RNA synthesis results in a 5'-poly(A) leader that allowed the unambiguous identification of true transcription start sites. Ninety RNA start sites were located just upstream of intermediate and late open reading frames, but many more appeared anomalous, occurring within coding and non-coding regions, indicating pervasive transcription initiation. We confirmed the presence of functional promoter sequences upstream of representative anomalous start sites and demonstrated that alternative start sites within open reading frames could generate truncated isoforms of proteins. In an analogous manner, poly(A) sequences allowed accurate mapping of the numerous 3'-ends of postreplicative RNAs, which were preceded by a pyrimidine-rich sequence in the DNA coding strand. The distribution of postreplicative promoter sequences throughout the genome provides enormous transcriptional complexity, and the large number of previously unmapped RNAs may have novel functions.

Conservation of a Triple-Helix-Forming RNA Stability Element in Noncoding and Genomic RNAs of Diverse Viruses

Abundant expression of the long noncoding (lnc) PAN (polyadenylated nuclear) RNA by the human oncogenic gammaherpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV) depends on a cis-element called the expression and nuclear retention element (ENE). The ENE upregulates PAN RNA by inhibiting its rapid nuclear decay through triple-helix formation with the poly(A) tail. Using structure-based bioinformatics, we identified six ENE-like elements in evolutionarily diverse viral genomes. Five are in double-stranded DNA viruses, including mammalian herpesviruses, insect polydnaviruses, and a protist mimivirus. One is in an insect picorna-like positive-strand RNA virus, suggesting that the ENE can counteract cytoplasmic as well as nuclear RNA decay pathways. Functionality of four of the ENEs was demonstrated by increased accumulation of an intronless polyadenylated reporter transcript in human cells. Identification of these ENEs enabled the discovery of PAN RNA homologs in two additional gammaherpesviruses, RRV and EHV2. Our findings demonstrate that searching for structural elements can lead to rapid identification of lncRNAs.

In vitro anti-viral effect of β-santalol against influenza viral replication

The anti-influenza A/HK (H3N2) virus activity of β-santalol was evaluated in MDCK cells and investigated the effect of β-santalol on synthesis of viral mRNAs. β-Santalol was investigated for its antiviral activity against influenza A/HK (H3N2) virus using a cytopathic effect (CPE) reduction method. β-Santalol exhibited anti-influenza A/HK (H3N2) virus activity of 86% with no cytotoxicity at the concentration of 100μg/ml reducing the formation of a visible CPE. Oseltamivir also showed moderate antiviral activity of about 83% against influenza A/HK (H3N2) virus at the concentration of 100μg/ml. Furthermore, the mechanism of anti-influenza virus action in the inhibition of viral mRNA synthesis was analyzed by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR), and the data indicated an inhibitory effect in late viral RNA synthesis compared with oseltamivir in the presence of 100μg/ml of β-santalol. β-Santalol should be further studied for therapeutic and prophylactic potential especially for influenza epidemics and pandemics.

Construction of a Full Transcription Map of Human Papillomavirus Type 18 during Productive Viral Infection

Human papillomavirus type 18 (HPV18) is the second most common oncogenic HPV genotype, responsible for ∼15% of cervical cancers worldwide. In this study, we constructed a full HPV18 transcription map using HPV18-infected raft tissues derived from primary human vaginal or foreskin keratinocytes. By using 5' rapid amplification of cDNA ends (RACE), we mapped two HPV18 transcription start sites (TSS) for early transcripts at nucleotide (nt) 55 and nt 102 and the HPV18 late TSS frequently at nt 811, 765, or 829 within the E7 open reading frame (ORF) of the virus genome. HPV18 polyadenylation cleavage sites for early and late transcripts were mapped to nt 4270 and mainly to nt 7299 or 7307, respectively, by using 3' RACE. Although all early transcripts were cleaved exclusively at a single cleavage site, HPV18 late transcripts displayed the heterogeneity of 3' ends, with multiple minor cleavage sites for late RNA polyadenylation. HPV18 splice sites/splice junctions for both early and late transcripts were identified by 5' RACE and primer walking techniques. Five 5' splice sites (donor sites) and six 3' splice sites (acceptor sites) that are highly conserved in other papillomaviruses were identified in the HPV18 genome. HPV18 L1 mRNA translates a L1 protein of 507 amino acids (aa), smaller than the 568 aa residues previously predicted. Collectively, a full HPV18 transcription map constructed from this report will lead us to further understand HPV18 gene expression and virus oncogenesis.

Silymarin efficacy against influenza A virus replication

The anti-influenza A/PR/8/34 virus activity of silymarin was evaluated in MDCK cells and investigated the effect of silymarin on synthesis of viral mRNAs. Silymarin was investigated for its antiviral activity against influenza A/PR virus using a cytopathic effect (CPE) reduction method. Silymarin exhibited anti-influenza A/PR/8/34 virus activity of 98% with no cytotoxicity at the concentration of 100 μg/ml reducing the formation of a visible CPE. Oseltamivir also did show moderate antiviral activity of about 52% against influenza A/PR/8/34 virus at the concentration of 100 μg/ml. Furthermore, the mechanism of anti-influenza virus action in the inhibition of viral mRNA synthesis was analyzed by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR), and the data indicated an inhibitory effect in late viral RNA synthesis compared with oseltamivir in the presence of 100 μg/ml of silymarin. Therefore, the potential of silymarin for use in treating influenza virus infection merits greater attention.

Transcriptional control in the prereplicative phase of T4 development

Control of transcription is crucial for correct gene expression and orderly development. For many years, bacteriophage T4 has provided a simple model system to investigate mechanisms that regulate this process. Development of T4 requires the transcription of early, middle and late RNAs. Because T4 does not encode its own RNA polymerase, it must redirect the polymerase of its host, E. coli, to the correct class of genes at the correct time. T4 accomplishes this through the action of phage-encoded factors. Here I review recent studies investigating the transcription of T4 prereplicative genes, which are expressed as early and middle transcripts. Early RNAs are generated immediately after infection from T4 promoters that contain excellent recognition sequences for host polymerase. Consequently, the early promoters compete extremely well with host promoters for the available polymerase. T4 early promoter activity is further enhanced by the action of the T4 Alt protein, a component of the phage head that is injected into E. coli along with the phage DNA. Alt modifies Arg265 on one of the two α subunits of RNA polymerase. Although work with host promoters predicts that this modification should decrease promoter activity, transcription from some T4 early promoters increases when RNA polymerase is modified by Alt. Transcription of T4 middle genes begins about 1 minute after infection and proceeds by two pathways: 1) extension of early transcripts into downstream middle genes and 2) activation of T4 middle promoters through a process called sigma appropriation. In this activation, the T4 co-activator AsiA binds to Region 4 of σ70, the specificity subunit of RNA polymerase. This binding dramatically remodels this portion of σ70, which then allows the T4 activator MotA to also interact with σ70. In addition, AsiA restructuring of σ70 prevents Region 4 from forming its normal contacts with the -35 region of promoter DNA, which in turn allows MotA to interact with its DNA binding site, a MotA box, centered at the -30 region of middle promoter DNA. T4 sigma appropriation reveals how a specific domain within RNA polymerase can be remolded and then exploited to alter promoter specificity.

Human Polyomavirus JC (JCV) Infection of Human B Lymphocytes: A Possible Mechanism for JCV Transmigration across the Blood‐Brain Barrier

It has been suggested that JC virus (JCV) might travel to the central nervous system in infected B cells. Moreover, recent data suggest the presence of JCV in bone marrow plasma cells. However, the evidence for infection and replication of JCV in B cells is unclear. To address this question, we infected Epstein-Barr virus-transformed B cells with JCV and found that the viral genome decreased >1000-fold from days 0 to 20 after infection, which concurred with the absence of viral early and late messenger RNA transcripts and proteins. However, immunofluorescent images of B cells infected with fluorescein isothiocyanate-conjugated JCV demonstrated that JCV enters the B cells, and DNase protection assay confirmed the presence of intact JCV virions inside the B cells. Moreover, JCV-infected B cells were able to transmit infection to naive glial cells. These data confirm that JCV nonproductively infects B cells and possibly uses them as a vehicle for transmigration across the blood-brain barrier.

Translation of intronless RNAs is strongly stimulated by the Epstein–Barr virus mRNA export factor EB2

The Epstein–Barr virus protein (EB2) allows the nuclear export of a particular subset of early and late viral RNAs derived from intronless genes. EB2 is conserved among most herpesvirus members and its presence is essential for the production of infectious particles. Here we show that, besides its role as a nuclear export factor, EB2 strongly stimulates translation of unspliced mRNAs without affecting overall cellular translation. Interestingly, this effect can be reversed by the addition of an intron within the gene. The spliced mRNA is then efficiently exported and translated even in the absence of EB2. Moreover, we show that EB2 associates with translating ribosomes and increases the proportion of its target RNA in the polyribosomal fraction. Finally, testing of EB2 homolog proteins derived from EBV-related herpesviruses, shows that, even if they play similar roles within the replication cycle of their respective virus, their mechanisms of action are different.

Gene regulation by sense–antisense overlap of polyadenylation signals

We show here that expression of genes from convergent transcription units can be regulated by the formation of double-stranded RNA (dsRNA) in the region of overlapping polyadenylation signals. The model system employed is the mouse polyomavirus. The early and late genes of polyomavirus are transcribed from opposite strands of the circular viral genome. At early times after infection, the early genes are expressed predominantly. Late gene expression increases dramatically upon the onset of DNA replication, when a major defect in polyadenylation of the late primary transcripts generates multigenomic RNAs that are precursors to the mature late mRNAs. Embedded in these late pre-mRNAs are sequences complementary to the early RNAs that act to down-regulate early gene expression via A-to-I editing of dsRNAs. In this system, the defective polyadenylation, and consequently the production of multigenomic late RNAs, depends on the context, and perhaps also, on the A-to-I editing of the poly(A) signal that overlaps the 3'-end of early transcripts.

Leucine aminopeptidase regulates defense and wound signaling in tomato downstream of jasmonic acid.

Leucine aminopeptidase A (LapA) is a late wound-response gene of tomato (Solanum lycopersicum). To elucidate the role of LapA, transgenic plants that overexpressed or abolished LapA gene expression were used. The early wound-response gene RNA levels were similar in wild-type and Lap-silenced (LapA-SI), -antisense (LapA-AS), and -overexpressing (LapA-OX) plants. By contrast, late wound-response gene RNA levels and protection against Manduca sexta damage were influenced by LapA RNA and protein levels. While LapA-OX plants had elevated levels of LapA RNAs and protein, ectopic expression of LapA was not sufficient to induce Pin (Ser proteinase inhibitor) or PPO (polyphenol oxidase) transcripts in nonwounded leaves. M. sexta larvae damaged less foliage and displayed delays in growth and development when feeding on LapA-OX plants. By contrast, LapA-SI and LapA-AS lines had lower levels of Pin and PPO RNAs than wild-type controls. Furthermore, larvae consumed more foliage and attained larger masses when feeding on LapA-SI plants. Jasmonic acid (JA) did not complement the wound-signaling phenotype of LapA-SI plants. Based on root elongation in the presence of JA, JA perception appeared to be intact in LapA-SI lines. Collectively, these data suggested that LAP-A has a role in modulating essential defenses against herbivores by promoting late wound responses and acting downstream of JA biosynthesis and perception.

Processing of a Membrane Protein Required for Cell-to-Cell Signaling during Endospore Formation in Bacillus subtilis

Activation of the late prespore-specific RNA polymerase sigma factor sigma(G) during Bacillus subtilis sporulation coincides with completion of the engulfment process, when the prespore becomes a protoplast fully surrounded by the mother cell cytoplasm and separated from it by a double membrane system. Activation of sigma(G) also requires expression of spoIIIJ, coding for a membrane protein translocase of the YidC/Oxa1p/Alb3 family, and of the mother cell-specific spoIIIA operon. Here we present genetic and biochemical evidence indicating that SpoIIIAE, the product of one of the spoIIIA cistrons, and SpoIIIJ interact in the membrane, thereby linking the function of the spoIIIJ and spoIIIA loci in the activation of sigma(G). We also show that SpoIIIAE has a functional Sec-type signal peptide, which is cleaved during sporulation. Furthermore, mutations that reduce or eliminate processing of the SpoIIIAE signal peptide arrest sporulation following engulfment completion and prevent activation of sigma(G). SpoIIIJ-type proteins can function in cooperation with or independently of the Sec system. In one model, SpoIIIJ interacts with SpoIIIAE in the context of the Sec translocon to promote its correct localization and/or topology in the membrane, so that it can signal the activation of sigma(G) following engulfment completion.