A unique two-gene gametophytic male sterility system in sorghum involving a possible role of RNA editing in fertility restoration. (49/4193)

The sorghum line IS1112C carries a male sterility-inducing cytoplasm when introduced into nuclear backgrounds that do not include fertility restoration genes. An mtDNA chimeric configuration resulting from recombination/duplication with atp9 resulted in the formation of orf107, a chimeric open reading frame. Transcription of orf107 is driven by three promoters, and abundant whole-length transcripts are detected in male-sterile lines. Fertility restoration is exacted through a unique two-gene gametophytic system requiring complementary action of genes designated Rf3 and Rf4. In male-sterile lines carrying Rf3, or lines restored to fertility, an enhanced nucleolytic transcript processing activity is targeted within orf107, cleaving 75% of whole-length transcripts. Rf3 thus confers or regulates the nucleolytic processing activity. A correlation between the frequency of RNA editing at two sites in orf107 and transcript processing suggests that processing may be dependent on templates edited at these sites. In addition, editing of atp6 transcripts is specifically reduced in anthers/pollen of male-sterile lines. Partially restored F1s and segregating F2s exhibit atp6 editing frequencies consistent with the possibility that Rf4 may confer the restitution of normal editing frequency. Thus RNA editing may be involved in features of fertility restoration in this unusual system.  (+info)

The N-terminal domain of Sxl protein disrupts Sxl autoregulation in females and promotes female-specific splicing of tra in males. (50/4193)

Sex determination in Drosophila depends upon the post-transcriptional regulatory activities of the Sex-lethal (Sxl) gene. Sxl maintains the female determined state and activates female differentiation pathways by directing the female-specific splicing of Sxl and tra pre-mRNAs. While there is compelling evidence that Sxl proteins regulate splicing by directly binding to target RNAs, previous studies indicate that the two Sxl RNA-binding domains are not in themselves sufficient for biological activity and that an intact N-terminal domain is also critical for splicing function. To further investigate the functions of the Sxl N terminus, we ectopically expressed a chimeric protein consisting of the N-terminal 99 amino acids fused to ss-galactosidase. The Nss-gal fusion protein behaves like a dominant negative, interfering with the Sxl autoregulatory feedback loop and killing females. This dominant negative activity can be attributed to the recruitment of the fusion protein into the large Sxl:Snf splicing complexes that are found in vivo and the consequent disruption of these complexes. In addition to the dominant negative activity, the Nss-gal fusion protein has a novel gain-of-function activity in males: it promotes the female-specific processing of tra pre-mRNAs. This novel activity is discussed in light of the blockage model for the tra splicing regulation.  (+info)

Cutting edge: silencing virus-specific cytotoxic T cell-mediated immune recognition by differential splicing: a novel implication of RNA processing for antigen presentation. (51/4193)

Persistent viruses have developed potent strategies to overcome host immune defenses. In particular, viral interference with Ag presentation by HLA class I molecules can effectively impair the host's CTL function. Here we provide evidence for a novel aspect of differential splicing on endogenous processing of a latent viral transcript resulting in dominant protein isoforms from which the CTL determinant has been deleted. Consequently, virus-infected cells expressing these isoforms were poorly recognized by CTLs. Molecular analysis revealed that this splicing significantly reduced expression of the viral transcript encoding the relevant epitope to levels below the threshold required for CTL recognition. The importance of splicing was further reinforced by the observation of efficient CTL recognition of target cells expressing a truncated viral transcript that abolished differential splicing. Thus, differential splicing, which is a common mechanism of gene regulation in many pathogens, may unexpectedly interfere with immune recognition.  (+info)

Specific transcriptional pausing activates polyadenylation in a coupled in vitro system. (52/4193)

We have developed a coupled in vitro transcription-polyadenylation system to investigate RNA polymerase II (Pol II) termination, which depends on active polyadenylation of the nascent RNA. Specific G-rich sequences originally identified as binding sites for the transcription factor MAZ both pause Pol II and activate polyadenylation of an upstream poly(A) signal. They do not affect polyadenylation efficiency in an uncoupled cleavage assay. In contrast, pausing of Pol II elongation induced by a high-affinity DNA-binding protein does not activate polyadenylation, indicating that G-rich MAZ sequences have a specific effect on polyadenylation. They also promote intrinsic pausing of purified Pol II, indicating a general role in the modulation of cotranscriptional RNA processing events.  (+info)

C-->U editing of apolipoprotein B mRNA in marsupials: identification and characterisation of APOBEC-1 from the American opossum Monodelphus domestica. (53/4193)

The C->U editing of RNA is widely found in plant and animal species. In mammals it is a discrete process confined to the editing of apolipoprotein B (apoB) mRNA in eutherians and the editing of the mitochondrial tRNA for glycine in marsupials. Here we have identified and characterised apoB mRNA editing in the American opossum Monodelphus domestica. The apoB mRNA editing site is highly conserved in the opossum and undergoes complete editing in the small intestine, but not in the liver or other tissues. Opossum APOBEC-1 cDNA was cloned, sequenced and expressed. The encoded protein is similar to APOBEC-1 of eutherians. Motifs previously identified as involved in zinc binding, RNA binding and catalysis, nuclear localisation and a C-terminal leucine-rich domain are all conserved. Opossum APOBEC-1 contains a seven amino acid C-terminal extension also found in humans and rabbits, but not present in rodents. The opossum APOBEC-1 gene has the same intron/exon organisation in the coding sequence as the eutherian gene. Northern blot and RT-PCR analyses and an editing assay indicate that no APOBEC-1 was expressed in the liver. Thus the far upstream promoter responsible for hepatic expression in rodents does not operate in the opossum. An APOBEC-1-like enzyme such as might be involved in C->U RNA editing of tRNA in marsupial mitochondria was not demonstrated. The activity of opossum APOBEC-1 in the presence of both chicken and rodent auxiliary editing proteins was comparable to that of other mammals. These studies extend the origins of APOBEC-1 back 170 000 000 years to marsupials and help bridge the gap in the origins of this RNA editing process between birds and eutherian mammals.  (+info)

Long 5' leaders inhibit removal of a 3' trailer from a precursor tRNA by mammalian tRNA 3' processing endoribonuclease. (54/4193)

Mammalian tRNA 3' processing endoribonuclease (3' tRNase) can remove a 3' trailer from various pre-tRNAs without 5' leader nucleotides. To examine how 5[prime] leader sequences affect 3' processing efficiency, we performed in vitro 3' processing reactions with purified pig 3' tRNase and pre-tRNAArgs containing a 13-nt 3' trailer and a 5[prime] leader of various lengths. The 3' processing was slightly stimulated by 5[prime] leaders containing up to 7 nt, whereas leaders of 9 nt or longer severely inhibited the reaction. Structure probing indicated that the 5' leader sequences had little effect on pre-tRNA folding. Similar results were obtained using pre-tRNA(Val)s containing a 5' leader of various lengths. We also investigated whether 3'tRNase can remove 3' trailers that are stably base-paired with 5' leaders to form an extended acceptor stem. Even such small 5' leaders as 3 and 6 nt, when base-paired with a 3' trailer, severely hindered removal of the 3' trailer by 3' tRNase.  (+info)

Assembly of 5S ribosomal RNA is required at a specific step of the pre-rRNA processing pathway. (55/4193)

A collection of yeast strains surviving with mutant 5S RNA has been constructed. The mutant strains presented alterations of the nucleolar structure, with less granular component, and a delocalization of the 25S rRNA throughout the nucleoplasm. The 5S RNA mutations affected helix I and resulted in decreased amounts of stable 5S RNA and of the ribosomal 60S subunits. The shortage of 60S subunits was due to a specific defect in the processing of the 27SB precursor RNA that gives rise to the mature 25S and 5.8S rRNA. The processing rate of the 27SB pre-rRNA was specifically delayed, whereas the 27SA and 20S pre-rRNA were processed at a normal rate. The defect was partially corrected by increasing the amount of mutant 5S RNA. We propose that the 5S RNA is recruited by the pre-60S particle and that its recruitment is necessary for the efficient processing of the 27SB RNA precursor. Such a mechanism could ensure that all newly formed mature 60S subunits contain stoichiometric amounts of the three rRNA components.  (+info)

Distinct export pathway utilized by the hepatitis B virus posttranscriptional regulatory element. (56/4193)

The posttranscriptional regulatory element (PRE) of hepatitis B virus is an RNA element important for the export of viral mRNA from the nucleus to the cytoplasm. The cellular export pathway utilized by the PRE is controversial. We present data showing that PRE-dependent export is blocked by vesicular stomatitis virus matrix protein, an inhibitor of all cellular RNA export other than tRNA export. It is also blocked by a mutated form of Ran-binding protein 1, which blocks export mediated by the human immunodeficiency virus Rev and Rev-response element (RRE) but not export mediated by the simian retrovirus constitutive transport element (CTE). On the other hand, PRE-dependent export is not blocked by either TAgRex or leptomycin B, two agents that prevent Rev/RRE-mediated export. Therefore, PRE appears to utilize an export pathway different from that of Rev/RRE or CTE.  (+info)