Heike Krebber

The iron-sulphur protein RNase L inhibitor functions in translation termination

The iron–sulphur (Fe–S)‐containing RNase L inhibitor (Rli1) is involved in ribosomal subunit maturation, transport of both ribosomal subunits to the cytoplasm, and translation initiation through interaction with the eukaryotic initiation factor 3 (eIF3) complex. Here, we present a new function for Rli1 in translation termination. Through co‐immunoprecipitation experiments, we show that Rli1 interacts physically with the translation termination factors eukaryotic release factor 1 (eRF1)/Sup45 and eRF3/Sup35 in Saccharomyces cerevisiae. Genetic interactions were uncovered between a strain depleted for Rli1 and sup35‐21 or sup45‐2. Furthermore, we show that downregulation of RLI1 expression leads to defects in the recognition of a stop codon, as seen in mutants of other termination factors. By contrast, RLI1 overexpression partly suppresses the read‐through defects in sup45‐2. Interestingly, we find that although the Fe–S cluster is not required for the interaction of Rli1 with eRF1 or its other interacting partner, Hcr1, from the initiation complex eIF3, it is required for its activity in translation termination; an Fe–S cluster mutant of RLI1 cannot suppress the read‐through defects of sup45‐2.

Yeast shuttling SR proteins Npl3p, Gbp2p, and Hrb1p are part of the translating mRNPs, and Npl3p can function as a translational repressor

ABSTRACT A major challenge in current molecular biology is to understand how sequential steps in gene expression are coupled. Recently, much attention has been focused on the linkage of transcription, processing, and mRNA export. Here we describe the cytoplasmic rearrangement for shuttling mRNA binding proteins in Saccharomyces cerevisiae during translation. While the bulk of Hrp1p, Nab2p, or Mex67p is not associated with polysome containing mRNAs, significant amounts of the serine/arginine (SR)-type shuttling mRNA binding proteins Npl3p, Gbp2p, and Hrb1p remain associated with the mRNA-protein complex during translation. Interestingly, a prolonged association of Npl3p with polysome containing mRNAs results in translational defects, indicating that Npl3p can function as a negative translational regulator. Consistent with this idea, a mutation in NPL3 that slows down translation suppresses growth defects caused by the presence of translation inhibitors or a mutation in eIF5A. Moreover, using sucrose density gradient analysis, we provide evidence that the import receptor Mtr10p, but not the SR protein kinase Sky1p, is involved in the timely regulated release of Npl3p from polysome-associated mRNAs. Together, these data shed light onto the transformation of an exporting to a translating mRNP.

Quality control of spliced mRNAs requires the shuttling SR proteins Gbp2 and Hrb1

Eukaryotic cells have to prevent the export of unspliced pre-mRNAs until intron removal is completed to avoid the expression of aberrant and potentially harmful proteins. Only mature mRNAs associate with the export receptor Mex67/TAP and enter the cytoplasm. Here we show that the two shuttling serine/arginine (SR)-proteins Gbp2 and Hrb1 are key surveillance factors for the selective export of spliced mRNAs in yeast. Their absence leads to the significant leakage of unspliced pre-mRNAs into the cytoplasm. They bind to pre-mRNAs and the spliceosome during splicing, where they are necessary for the surveillance of splicing and the stable binding of the TRAMP complex to spliceosome-bound transcripts. Faulty transcripts are marked for their degradation at the nuclear exosome. On correct mRNAs the SR proteins recruit Mex67 upon completion of splicing to allow a quality controlled nuclear export. Altogether, these data identify a role for shuttling SR proteins in mRNA surveillance and nuclear mRNA quality control.

Identification of Gbp2 as a novel poly(A)+ RNA-binding protein involved in the cytoplasmic delivery of messenger RNAs in yeast

Important progress in understanding messenger RNA export from the nucleus could be achieved by increasing the list of proteins that are involved in this process. Here, we present the identification of Gbp2 as a novel shuttling RNA‐binding protein in Saccharomyces cerevisiae. Nuclear import of Gbp2 is dependent on the receptor Mtr10 and the serine/arginine‐specific protein kinase Sky1. Deletion of the genes encoding both of these proteins or disruption of two of the arginine/serine repeats each shifts the steady‐state localization of Gbp2 to the cytoplasm. Interestingly, deletion of MTR10 only also causes an increase in poly(A)+ RNA binding by Gbp2, suggesting a role of Mtr10 in the dissociation of Gbp2 from mRNA in the cytoplasm. The nuclear export of Gbp2 is always coupled to mRNA export and is dependent on continuous RNA polymerase II transcription and mRNA‐export factors. Although GBP2 is not essential for normal cell growth, overexpression of this gene is toxic and causes a nuclear retention of bulk poly(A)+ RNA. Together, our findings clearly show an involvement of Gbp2 in mRNA transport. In addition, as a non‐essential protein, Gbp2 also has the interesting potential to be spatially or temporally regulated.

Differential Export Requirements for Shuttling Serine/Arginine-type mRNA-binding Proteins

Messenger RNAs are transported to the cytoplasm bound to several shuttling mRNA-binding proteins. Here, we present the characterization of Hrb1, a novel component of the transported ribonucleoprotein complex in Saccharomyces cerevisiae. The protein is similar to the other two serine/arginine (SR)-type proteins in yeast, Gbp2 and Npl3. Hrb1 is nuclear at steady state and its import is mediated by the karyopherin Mtr10. Hrb1 binds to poly(A)+ RNA in vivo and its binding is significantly increased in MTR10 mutants, suggesting a role for Mtr10 in dissociating Hrb1 from the mRNAs. Interestingly, by comparing the export requirements of all three SR proteins we find similarities but also striking differences. While the export of all three proteins is dependent on the export of mRNAs in general, as no transport is observed in mutants defective in transcription (rpb1-1) or mRNA export (mex67-5), we find specific requirements for components of the THO complex, involved in transcription elongation. While both Hrb1 and Gbp2 depend on Mft1 and Hpr1 for their nuclear export, Npl3 is exported independently of both proteins. These findings suggest that Hrb1 and Gbp2, but not Npl3, might be loaded onto the growing mRNA via the THO complex components Mtf1 and Hrp1.

DEAD-Box RNA Helicases in Bacillus subtilis Have Multiple Functions and Act Independently from Each Other

DEAD-box RNA helicases play important roles in remodeling RNA molecules and in facilitating a variety of RNA-protein interactions that are key to many essential cellular processes. In spite of the importance of RNA, our knowledge about RNA helicases is limited. In this study, we investigated the role of the four DEAD-box RNA helicases in the Gram-positive model organism Bacillus subtilis. A strain deleted of all RNA helicases is able to grow at 37°C but not at lower temperatures. The deletion of cshA, cshB, or yfmL in particular leads to cold-sensitive phenotypes. Moreover, these mutant strains exhibit unique defects in ribosome biogenesis, suggesting distinct functions for the individual enzymes in this process. Based on protein accumulation, severity of the cold-sensitive phenotype, and the interaction with components of the RNA degradosome, CshA is the major RNA helicase of B. subtilis. To unravel the functions of CshA in addition to ribosome biogenesis, we conducted microarray analysis and identified the ysbAB and frlBONMD mRNAs as targets that are strongly affected by the deletion of the cshA gene. Our findings suggest that the different helicases make distinct contributions to the physiology of B. subtilis. Ribosome biogenesis and RNA degradation are two of their major tasks in B. subtilis.

The mRNA export factor Npl3 mediates the nuclear export of large ribosomal subunits

The nuclear export of large ribonucleoparticles is complex and requires specific transport factors. Messenger RNAs are exported through the RNA‐binding protein Npl3 and the interacting export receptor Mex67. Export of large ribosomal subunits also requires Mex67; however, in this case, Mex67 binds directly to the 5S ribosomal RNA (rRNA) and does not require the Npl3 adaptor. Here, we have discovered a new function of Npl3 in mediating the export of pre‐60S ribosomal subunit independently of Mex67. Npl3 interacts with the 25S rRNA, ribosomal and ribosome‐associated proteins, as well as with the nuclear pore complex. Mutations in NPL3 lead to export defects of the large subunit and genetic interactions with other pre‐60S export factors.

Dbp5 - from nuclear export to translation.

The DEAD-box RNA helicase Dbp5 is an essential and conserved mRNA export factor which functions in the ATP dependent remodeling of RNA/protein complexes. As such it displaces mRNA bound proteins at the cytoplasmic site of the nuclear pore complex. For the regulation of its RNA-dependent ATPase activity during late steps of nuclear transport, Dbp5 requires the nucleoporin Nup159 and its cofactors Gle1 and IP6. In addition to its role in mRNA export, a second important function of Dbp5 was identified in translation termination, where it acts together with eRF1 once the translation machinery has reached the stop codon. Similar to mRNA export, this function also requires Gle1-IP6, however, the counterpart of Nup159 is still missing. Potential other functions of the nucleo-cytoplasmic protein Dbp5 are discussed as well as its substrate specificity and details in its regulatory cycle that are based on recent biochemical and structural characterization. This article is part of a Special Issue entitled: The Biology of RNA helicases - Modulation for life.

A Subset of Histone H2B Genes Produces Polyadenylated mRNAs under a Variety of Cellular Conditions

Unlike other metazoan mRNAs, replication-dependent histone gene transcripts are not polyadenylated but instead have a conserved stem-loop structure at their 3′ end. Our previous work has shown that under certain conditions replication-dependent histone genes can produce alternative transcripts that are polyadenylated at the 3′ end and, in some cases, spliced. A number of microarray studies examining the expression of polyadenylated mRNAs identified changes in the levels of histone transcripts e.g. during differentiation and tumorigenesis. However, it remains unknown which histone genes produce polyadenylated transcripts and which conditions regulate this process. In the present study we examined the expression and polyadenylation of the human histone H2B gene complement in various cell lines. We demonstrate that H2B genes display a distinct expression pattern that is varies between different cell lines. Further we show that the fraction of polyadenylated HIST1H2BD and HIST1H2AC transcripts is increased during differentiation of human mesenchymal stem cells (hMSCs) and human fetal osteoblast (hFOB 1.19). Furthermore, we observed an increased fraction of polyadenylated transcripts produced from the histone genes in cells following ionizing radiation. Finally, we show that polyadenylated transcripts are transported to the cytoplasm and found on polyribosomes. Thus, we propose that the production of polyadenylated histone mRNAs from replication-dependent histone genes is a regulated process induced under specific cellular circumstances.

mRNA quality control is bypassed for immediate export of stress-responsive transcripts.

Cells grow well only in a narrow range of physiological conditions. Surviving extreme conditions requires the instantaneous expression of chaperones that help to overcome stressful situations. To ensure the preferential synthesis of these heat-shock proteins, cells inhibit transcription, pre-mRNA processing and nuclear export of non-heat-shock transcripts, while stress-specific mRNAs are exclusively exported and translated. How cells manage the selective retention of regular transcripts and the simultaneous rapid export of heat-shock mRNAs is largely unknown. In Saccharomyces cerevisiae, the shuttling RNA adaptor proteins Npl3, Gbp2, Hrb1 and Nab2 are loaded co-transcriptionally onto growing pre-mRNAs. For nuclear export, they recruit the export-receptor heterodimer Mex67–Mtr2 (TAP–p15 in humans). Here we show that cellular stress induces the dissociation of Mex67 and its adaptor proteins from regular mRNAs to prevent general mRNA export. At the same time, heat-shock mRNAs are rapidly exported in association with Mex67, without the need for adapters. The immediate co-transcriptional loading of Mex67 onto heat-shock mRNAs involves Hsf1, a heat-shock transcription factor that binds to heat-shock-promoter elements in stress-responsive genes. An important difference between the export modes is that adaptor-protein-bound mRNAs undergo quality control, whereas stress-specific transcripts do not. In fact, regular mRNAs are converted into uncontrolled stress-responsive transcripts if expressed under the control of a heat-shock promoter, suggesting that whether an mRNA undergoes quality control is encrypted therein. Under normal conditions, Mex67 adaptor proteins are recruited for RNA surveillance, with only quality-controlled mRNAs allowed to associate with Mex67 and leave the nucleus. Thus, at the cost of error-free mRNA formation, heat-shock mRNAs are exported and translated without delay, allowing cells to survive extreme situations.