Dataset Information

Stem-loop RNA labeling can affect nuclear and cytoplasmic mRNA processing.

ABSTRACT: The binding of sequence-specific RNA-interacting proteins, such as the bacteriophage MS2 or PP7 coat proteins, to their corresponding target sequences has been extremely useful and widely used to visualize single mRNAs in vivo. However, introduction of MS2 stem-loops into yeast mRNAs has recently been shown to lead to the accumulation of RNA fragments, suggesting that the loops impair mRNA decay. This result was questioned, because fragment occurrence was mainly assessed using ensemble methods, and their cellular localization and its implications had not been addressed on a single transcript level. Here, we demonstrate that the introduction of either MS2 stem-loops (MS2SL) or PP7 stem-loops (PP7SL) can affect the processing and subcellular localization of mRNA. We use single-molecule fluorescence in situ hybridization (smFISH) to determine the localization of three independent mRNAs tagged with the stem-loop labeling systems in glucose-rich and glucose starvation conditions. Transcripts containing MS2SL or PP7SL display aberrant localization in both the nucleus and the cytoplasm. These defects are most prominent in glucose starvation conditions, with nuclear mRNA processing being altered and stem-loop fragments abnormally enriching in processing bodies (PBs). The mislocalization of SL-containing RNAs is independent of the presence of the MS2 or PP7 coat protein (MCP or PCP).

SUBMITTER: Heinrich S

PROVIDER: S-EPMC5238788 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:BackgroundAnaerobic, mesophilic, and cellulolytic Ruminiclostridium cellulolyticum produces an efficient cellulolytic extracellular complex named cellulosome, which consist of a non-catalytic multi-functional integrating subunit, organizing the various catalytic subunits into the complex. Main components of cellulosome were encoded by the cip-cel operon in R. cellulolyticum, and their stoichiometry is controlled by the mechanism of selective RNA processing and stabilization, which allows to confer each processed RNA portion from the cip-cel mRNA on different fates due to their stability and resolve the potential contradiction between the equimolar stoichiometry of transcripts with a within a transcription unit and the non-equimolar stoichiometry of subunits.ResultsIn this work, RNA processing events were found to occur at six intergenic regions (IRs) harboring stem-loop structures in cip-cel operon. These stem-loops not only stabilize processed transcripts at their both ends, but also act as cleavage signals specifically recognized by endoribonucleases. We further demonstrated that cleavage sites were often located downstream or 3' end of their associated stem-loops that could be classified into two types, with distinct GC-rich stems being required for RNA cleavage. However, the cleavage site in IR4 was found to be located upstream of the stem-loop, as determined by the bottom AT-pair region of this stem-loop, together with its upstream structure. Thus, our findings reveal the structural requirements for processing of cip-cel transcripts, which can be potentially used to control the stoichiometry of gene expression in an operon.ConclusionsOur findings reveal that stem-loop structures acting as RNA cleavage signals not only can be recognized by endoribonucleases and determine the location of cleavage sites but also determine the stoichiometry of their flanking processed transcripts by controlling stability in cip-cel operon. These features represent a complexed regulation of cellulosome in the post-transcriptional level, which can be exploited for designing synthetic elements to control gene expression.

Dataset Information

Stem-loop RNA labeling can affect nuclear and cytoplasmic mRNA processing.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets