Small RNAs derived from longer non-coding RNAs.

Posttranscriptional gene regulation by small RNAs and its crucial impact on development, apoptosis, stem cell self-renewal and differentiation gained tremendous scientific attention since the discovery of RNA interference (RNAi) and microRNAs (miRNAs). However, in the last few years, many more examples for regulatory small RNAs were discovered, some of them even with miRNA-like functions. Even though these small RNA molecules were previously thought to be mere artifacts accumulating during the preparation of RNA libraries, advances in sequencing technology revealed that small RNAs derive from hairpin-fold RNA structures, for example. Mirtrons, short hairpin RNAs or small RNAs that are processed from longer non-coding RNAs such as tRNAs or snoRNAs have been found recently and some of them might be involved in the regulation of gene expression in different organisms. Furthermore, small RNAs originating from transposable elements, heterochromatic regions or convergent transcription units forming endogenous short interfering RNAs (endo-siRNAs) are the somatic equivalents of the germline-specific Piwi-interacting RNAs (piRNAs) in mediating transposon silencing. This review will focus on several recent findings that have added new aspects to small RNA-guided gene silencing.

Nucleocytoplasmic shuttling of the La motif-containing protein Sro9 might link its nuclear and cytoplasmic functions.

Diverse steps in gene expression are tightly coupled. Curiously, the La-motif-containing protein Sro9 has been shown to play a role in transcription and translation. Here, we show that Sro9 interacts with nuclear and cytoplasmic protein complexes involved in gene expression. In addition, Sro9 shuttles between nucleus and cytoplasm and is exported from the nucleus in an mRNA export-dependent manner. Importantly, Sro9 is recruited to transcribed genes. However, whole genome expression analysis shows that loss of Sro9 function does not greatly change the level of specific transcripts indicating that Sro9 does not markedly affect their synthesis and/or stability. Taken together, Sro9 might bind to the mRNP already during transcription and accompany the mature mRNP to the cytoplasm where it modulates translation of the mRNA.

Structure-system correlation identifies a gene regulatory Mediator submodule.

A combination of crystallography, biochemistry, and gene expression analysis identifies the coactivator subcomplex Med8C/18/20 as a functionally distinct submodule of the Mediator head module. Med8C forms a conserved alpha-helix that tethers Med18/20 to the Mediator. Deletion of Med8C in vivo results in dissociation of Med18/20 from Mediator and in loss of transcription activity of extracts. Deletion of med8C, med18, or med20 causes similar changes in the yeast transcriptome, establishing Med8C/18/20 as a predominantly positive, gene-specific submodule required for low transcription levels of nonactivated genes, including conjugation genes. The presented structure-based system perturbation is superior to gene deletion analysis of gene regulation.

The RNA polymerase II CTD kinase Ctk1 functions in translation elongation.

Translation is a highly complex process that is regulated by a multitude of factors. Here, we show that the conserved kinase Ctk1 functions in translation by enhancing decoding fidelity. Ctk1 associates with translating ribosomes in vivo and is needed for efficient translation. Ctk1 phosphorylates Rps2, a protein of the small ribosomal subunit, on Ser 238. Importantly, Ctk1-depleted as well as rps2-S238A mutant cells show a defect in translation elongation through an increase in the frequency of miscoding. The role of Ctk1 in translation may be conserved as the mammalian homolog of Ctk1, CDK9, also associates with polysomes. Since Ctk1 interacts with the TREX (transcription and mRNA export) complex, which couples transcription to mRNA export, Ctk1/CDK9 might bind to correctly processed mRNPs during transcription and accompany the mRNP to the ribosomes in the cytoplasm, where Ctk1 enhances efficient and accurate translation of the mRNA.

A missense mutation in the 3-ketodihydrosphingosine reductase FVT1 as candidate causal mutation for bovine spinal muscular atrophy.

The bovine form of the autosomal recessive neurodegenerative disease spinal muscular atrophy (SMA) shows striking similarity to the human form of the disease. It has, however, been mapped to a genomic region not harboring the bovine orthologue of the SMN gene, mutation of which causes human SMA. After refinement of the mapping results we analyzed positional and functional candidate genes. One of three candidate genes, FVT1, encoding 3-ketodihydrosphingosine reductase, which catalyzes a crucial step in the glycosphingolipid metabolism, showed a G-to-A missense mutation that changes Ala-175 to Thr. The identified mutation is limited to SMA-affected animals and carriers and always appears in context of the founder haplotype. The Ala variant found in healthy animals showed the expected 3-ketodihydrosphingosine reductase activity in an in vitro enzyme assay. Importantly, the Thr variant found in SMA animals showed no detectable activity. Surprisingly, in an in vivo assay the mutated gene complements the growth defect of a homologous yeast knockout strain as well as the healthy variant. This finding explains the viability of affected newborn calves and the later neuron-specific onset of the disease, which might be due to the high sensitivity of these neurons to changes in housekeeping functions. Taken together, the described mutation in FVT1 is a strong candidate for causality of SMA in cattle. This result provides an animal model for understanding the underlying mechanisms of the development of SMA and will allow efficient selection against the disease in cattle.

Swt1, a novel yeast protein, functions in transcription.

The conserved TREX complex couples transcription to nuclear mRNA export. Here, we report that the uncharacterized open reading frame YOR166c genetically interacts with TREX complex components and encodes a novel protein named Swt1 for "synthetically lethal with TREX." Co-immunoprecipitation experiments show that Swt1 also interacts with the TREX complex biochemically. Consistent with a potential role in transcription as suggested by its interaction with TREX, Swt1 localizes mainly to the nucleus. Importantly, deletion of Swt1 leads to decreased transcription. Taken together, these data suggest that Swt1 functions in gene expression in conjunction with the TREX complex.

Structure and TBP binding of the Mediator head subcomplex Med8-Med18-Med20.

The Mediator head module stimulates basal RNA polymerase II (Pol II) transcription and enables transcriptional regulation. Here we show that the head subunits Med8, Med18 and Med20 form a subcomplex (Med8/18/20) with two submodules. The highly conserved N-terminal domain of Med8 forms one submodule that binds the TATA box-binding protein (TBP) in vitro and is essential in vivo. The second submodule consists of the C-terminal region of Med8 (Med8C), Med18 and Med20. X-ray analysis of this submodule reveals that Med18 and Med20 form related beta-barrel folds. A conserved putative protein-interaction face on the Med8C/18/20 submodule includes sites altered by srb mutations, which counteract defects resulting from Pol II truncation. Our results and published data support a positive role of the Med8/18/20 subcomplex in initiation-complex formation and suggest that the Mediator head contains a multipartite TBP-binding site that can be modulated by transcriptional activators.

Cotranscriptional recruitment of the serine-arginine-rich (SR)-like proteins Gbp2 and Hrb1 to nascent mRNA via the TREX complex.

The TREX (transcription/export) complex couples transcription elongation to the nuclear export of mRNAs. In this article, we show that the poly(A)(+) RNA-binding proteins Gbp2 and Hrb1, which resemble the serine-arginine-rich (SR) family of splicing factors found in higher eukaryotes, are specifically associated with the yeast TREX complex. We also show that Gbp2 and Hrb1 interact with Ctk1, a kinase that phosphorylates the C-terminal domain of RNA polymerase II during transcription elongation. Consistent with these findings, Gbp2 and Hrb1 associate with actively transcribed genes throughout their entire lengths. By using an RNA immunoprecipitation assay, we show that Gbp2 and Hrb1 also are bound to transcripts that are derived from these genes. We conclude that recruitment of the SR-like proteins Gbp2 and Hrb1 to mRNA occurs cotranscriptionally by means of association with the TREX complex and/or Ctk1.

A novel type of replicative enzyme harbouring ATPase, primase and DNA polymerase activity.

Although DNA replication is a process common in all domains of life, primase and replicative DNA polymerase appear to have evolved independently in the bacterial domain versus the archaeal/eukaryal branch of life. Here, we report on a new type of replication protein that constitutes the first member of the DNA polymerase family E. The protein ORF904, encoded by the plasmid pRN1 from the thermoacidophile archaeon Sulfolobus islandicus, is a highly compact multifunctional enzyme with ATPase, primase and DNA polymerase activity. Recombinant purified ORF904 hydrolyses ATP in a DNA-dependent manner. Deoxynucleotides are preferentially used for the synthesis of primers approximately 8 nucleotides long. The DNA polymerase activity of ORF904 synthesizes replication products of up to several thousand nucleotides in length. The primase and DNA polymerase activity are located in the N-terminal half of the protein, which does not show homology to any known DNA polymerase or primase. ORF904 constitutes a new type of replication enzyme, which could have evolved independently from the eubacterial and archaeal/eukaryal proteins of DNA replication.