Deletion of the Sm1 encoding motif in the lsm gene results in distinct changes in the transcriptome and enhanced swarming activity of Haloferax cells

Abstract

Members of the Sm protein family are important for the cellular RNA metabolism in all three domains of life. The family includes archaeal and eukaryotic Lsm proteins, eukaryotic Sm proteins and archaeal and bacterial Hfq proteins. While several studies concerning the bacterial and eukaryotic family members have been published, little is known about the archaeal Lsm proteins. Although structures for several archaeal Lsm proteins have been solved already more than ten years ago, we still do not know much about their biological function, however one can confidently propose that the archaeal Lsm proteins will also be involved in RNA metabolism. Therefore, we investigated this protein in the halophilic archaeon Haloferax volcanii. The Haloferax genome encodes a single Lsm protein, the lsm gene overlaps and is co-transcribed with the gene for the ribosomal L37.eR protein. Here, we show that the reading frame of the lsm gene contains a promoter which regulates expression of the overlapping rpl37R gene. This rpl37R specific promoter ensures high expression of the rpl37R gene in exponential growth phase. To investigate the biological function of the Lsm protein we generated a lsm deletion mutant that had the coding sequence for the Sm1 motif removed but still contained the internal promoter for the downstream rpl37R gene. The transcriptome of this deletion mutant was compared to the wild type transcriptome, revealing that several genes are down-regulated and many genes are up-regulated in the deletion strain. Northern blot analyses confirmed down-regulation of two genes. In addition, the deletion strain showed a gain of function in swarming, in congruence with the up-regulation of transcripts encoding proteins required for motility.

The SmAP1/2 proteins of the crenarchaeon Sulfolobus solfataricus interact with the exosome and stimulate A-rich tailing of transcripts

“…In addition, the Hv SmAP was shown to co-purify with several uncharacterized non-coding RNAs, tRNAs and C/D box snoRNAs ( 28 ). A deletion of the Sm1 motif in the Hv SmAP encoding gene showed a gain of function in swarming, which agreed with the up-regulation of transcripts encoding proteins required for motility ( 32 ).…”

Identification of RNA 3´ ends and termination sites in Haloferax volcanii

“…In addition, the upstream lsm gene can be terminated within the downstream rpl37e gene. A dicistronic transcript and a rpl37e mRNA were identified by northern blot analysis [30]. A similar independent transcription of genes that are part of the same operon has been described in Sulfolobus islandicus [31].…”

“…This phenomenon was confirmed in a previous study experimentally, for instance for the dicistronic operon encoding the proteins Lsm and L37e (HVO_2723 and HVO_2722). Here, a promoter for expression of the rpl37e gene is located in the upstream lsm gene (Supplementary Figure 5) [30]. In addition, the upstream lsm gene can be terminated within the downstream rpl37e gene.…”

“…To allow the use of a plasmid carrying the ß-galactosidase reporter gene (a fusion of ß-galactosidase genes from Haloferax alicante and Haloferax volcanii)(bgaHa) [50,51], the H. volcanii ßgalactosidase gene (bgaH) was deleted in strain H119. H119 was transformed with the bgaH deletion plasmid pTA617 (for a list of plasmids used see Supplementary Table 5) [30,52] and grown in Hv-Ca medium with tryptophan (final concentration 0.25 mM) to generate the deletion strain [53]. Homozygous knock-out clones were verified via southern-blot using SalI digested genomic DNA and probe bgaHaDO (primers: bgaHKODO-for and bgaHKODO-rev (for a list of primers used see Supplementary Table 5); template genomic DNA of H. volcanii).…”

The SmAP2 RNA binding motif in the 3′UTR affects mRNA stability in the crenarchaeum Sulfolobus solfataricus

“…Another study showed that the Haloferax volcanii (Hv) Sm protein binds to several uncharacterized ncRNAs, tRNAs and C/D box snoRNAs ( 18 ), again suggesting a function in tRNA/rRNA processing. A deletion of the Sm1 motif in the Hv SmAP1 encoding gene showed a gain of function in swarming, which was paralleled by an up-regulation of transcripts encoding proteins required for motility ( 24 ).…”