scholarly journals Molecular Determinants for RNA Release into Extracellular Vesicles

Cells ◽  
2021 ◽  
Vol 10 (10) ◽  
pp. 2674
Author(s):  
Marie-Luise Mosbach ◽  
Christina Pfafenrot ◽  
Elke Pogge von Strandmann ◽  
Albrecht Bindereif ◽  
Christian Preußer
Keyword(s):  
Extracellular Vesicles ◽  
Rna Polymerase Iii ◽  
Bound State ◽  
Reporter System ◽  
U6 Snrna ◽  
Copy Numbers ◽  
Gapdh Mrna ◽  
Sequence Elements ◽  
Rna Accumulation ◽  
Pol Iii

Extracellular vesicles (EVs) are important for intercellular communication and act as vehicles for biological material, such as various classes of coding and non-coding RNAs, a few of which were shown to selectively target into vesicles. However, protein factors, mechanisms, and sequence elements contributing to this specificity remain largely elusive. Here, we use a reporter system that results in different types of modified transcripts to decipher the specificity determinants of RNAs released into EVs. First, we found that small RNAs are more efficiently packaged into EVs than large ones, and second, we determined absolute quantities for several endogenous RNA transcripts in EVs (U6 snRNA, U1 snRNA, Y1 RNA, and GAPDH mRNA). We show that RNA polymerase III (pol III) transcripts are more efficiently secreted into EVs compared to pol II-derived transcripts. Surprisingly, our quantitative analysis revealed no RNA accumulation in the vesicles relative to the total cellular levels, based on both overexpressed reporter transcripts and endogenous RNAs. RNA appears to be EV-associated only at low copy numbers, ranging between 0.02 and 1 molecule per EV. This RNA association may reflect internal EV encapsulation or a less tightly bound state at the vesicle surface.

scholarly journals Assembly of SNAPc, Bdp1, and TBP on the U6 snRNA Gene Promoter in Drosophila melanogaster

2020 ◽  
Vol 40 (12) ◽  
Author(s):  
Mun Kyoung Kim ◽  
An Tranvo ◽  
Ann Marie Hurlburt ◽  
Neha Verma ◽  
Phuc Phan ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Drosophila Melanogaster ◽  
Rna Polymerase Iii ◽  
Gene Promoter ◽  
Stable Complex ◽  
Sequence Element ◽  
Content Type ◽  
U6 Snrna ◽  
U6 Promoter ◽  
Pol Iii

ABSTRACT U6 snRNA is transcribed by RNA polymerase III (Pol III) and has an external upstream promoter that consists of a TATA sequence recognized by the TBP subunit of the Pol III basal transcription factor IIIB and a proximal sequence element (PSE) recognized by the small nuclear RNA activating protein complex (SNAPc). Previously, we found that Drosophila melanogaster SNAPc (DmSNAPc) bound to the U6 PSE can recruit the Pol III general transcription factor Bdp1 to form a stable complex with the DNA. Here, we show that DmSNAPc-Bdp1 can recruit TBP to the U6 promoter, and we identify a region of Bdp1 that is sufficient for TBP recruitment. Moreover, we find that this same region of Bdp1 cross-links to nucleotides within the U6 PSE at positions that also cross-link to DmSNAPc. Finally, cross-linking mass spectrometry reveals likely interactions of specific DmSNAPc subunits with Bdp1 and TBP. These data, together with previous findings, have allowed us to build a more comprehensive model of the DmSNAPc-Bdp1-TBP complex on the U6 promoter that includes nearly all of DmSNAPc, a portion of Bdp1, and the conserved region of TBP.

scholarly journals Chromatin Structure and Expression of a Gene Transcribed by RNA Polymerase III Are Independent of H2A.Z Deposition

2008 ◽  
Vol 28 (8) ◽  
pp. 2598-2607 ◽  
Author(s):  
Aneeshkumar Gopalakrishnan Arimbasseri ◽  
Purnima Bhargava
Keyword(s):  
Rna Polymerase ◽  
Chromatin Structure ◽  
Rna Polymerase Iii ◽  
Nutritional Deprivation ◽  
U6 Snrna ◽  
Polymerase Iii ◽  
Downstream Box ◽  
Pol Iii

ABSTRACT The genes transcribed by RNA polymerase III (Pol III) generally have intragenic promoter elements. One of them, the yeast U6 snRNA (SNR6) gene is activated in vitro by a positioned nucleosome between its intragenic box A and extragenic, downstream box B separated by ∼200 bp. We demonstrate here that the in vivo chromatin structure of the gene region is characterized by the presence of an array of positioned nucleosomes, with only one of them in the 5′ end of the gene having a regulatory role. A positioned nucleosome present between boxes A and B in vivo does not move when the gene is repressed due to nutritional deprivation. In contrast, the upstream nucleosome which covers the TATA box under repressed conditions is shifted ∼50 bp further upstream by the ATP-dependent chromatin remodeler RSC upon activation. It is marked with the histone variant H2A.Z and H4K16 acetylation in active state. In the absence of H2A.Z, the chromatin structure of the gene does not change, suggesting that H2A.Z is not required for establishing the active chromatin structure. These results show that the chromatin structure directly participates in regulation of a Pol III-transcribed gene under different states of its activity in vivo.

Recent insights into regulation of transcription by RNA polymerase III and the cellular functions of its transcripts

2011 ◽  
Vol 392 (5) ◽  
Author(s):  
Tatyana V. Nikitina ◽  
Lyudmila I. Tischenko ◽  
Wolfgang A. Schulz
Keyword(s):  
Rna Polymerase ◽  
Stress Responses ◽  
Rna Polymerase Iii ◽  
Cell Types ◽  
Regulation Of Transcription ◽  
Class Iii ◽  
Cellular Functions ◽  
U6 Snrna ◽  
Polymerase Iii ◽  
Pol Iii

AbstractThe products of transcription by the multisubunit enzyme RNA polymerase III (Pol III), such as 5S rRNA, tRNAs, U6 snRNA, are important for cell growth, proliferation and differentiation. The known range of the Pol III transcriptome has expanded over recent years, and novel functions of the newly discovered and already well known transcripts have been identified, including regulation of stress responses and apoptosis. Furthermore, transcription by Pol III has turned out to be strongly regulated, differing between diverse class III genes, among cell types and under stress conditions. The mechanisms involved in regulation of Pol III transcription are being elucidated and disturbances in that regulation have been implicated in various diseases, including cancer. This review summarizes the novel data on the regulation of RNA polymerase III transcription, including epigenetic and gene specific mechanisms and outlines recent insights into the cellular functions of the Pol III transcriptome, in particular of SINE RNAs.

scholarly journals High-Level Activation of Transcription of the Yeast U6 snRNA Gene in Chromatin by the Basal RNA Polymerase III Transcription Factor TFIIIC

2004 ◽  
Vol 24 (9) ◽  
pp. 3596-3606 ◽  
Author(s):  
Sushma Shivaswamy ◽  
George A. Kassavetis ◽  
Purnima Bhargava
Keyword(s):  
Transcription Factor ◽  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Direct Role ◽  
U6 Snrna ◽  
Snrna Gene ◽  
Polymerase Iii ◽  
Activation Of Transcription ◽  
Pol Iii

ABSTRACT Transcription of the U6 snRNA gene (SNR6) in Saccharomyces cerevisiae by RNA polymerase III (pol III) requires TFIIIC and its box A and B binding sites. In contrast, TFIIIC has little or no effect on SNR6 transcription with purified components in vitro due to direct recognition of the SNR6 TATA box by TFIIIB. When SNR6 was assembled into chromatin in vitro by use of the Drosophila melanogaster S-190 extract, transcription of these templates with highly purified yeast pol III, TFIIIC, and TFIIIB displayed a near-absolute requirement for TFIIIC but yielded a 5- to 15-fold-higher level of transcription relative to naked DNA (>100-fold activation over repressed chromatin). Analysis of chromatin structure demonstrated that TFIIIC binding leads to remodeling of U6 gene chromatin, resulting in positioning of a nucleosome between boxes A and B. The resulting folding of the intervening DNA into the nucleosome could bring the suboptimally spaced SNR6 box A and B elements into greater proximity and thus facilitate activation of transcription. In the absence of ATP, however, the binding of TFIIIC to box B in chromatin was not accompanied by remodeling and the transcription activation was ∼35% of that seen in its presence, implying that both TFIIIC binding and ATP-dependent chromatin remodeling were required for the full activation of the gene. Our results suggest that TFIIIC, which is a basal transcription factor of pol III, also plays a direct role in remodeling chromatin on the SNR6 gene.

scholarly journals Reprogramming mRNA Expression in Response to Defect in RNA Polymerase III Assembly in the Yeast Saccharomyces cerevisiae

2021 ◽  
Vol 22 (14) ◽  
pp. 7298
Author(s):  
Izabela Rudzińska ◽  
Małgorzata Cieśla ◽  
Tomasz W. Turowski ◽  
Alicja Armatowska ◽  
Ewa Leśniewska ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Ribosome Biogenesis ◽  
Rna Polymerase Iii ◽  
Mrna Levels ◽  
Rna Seq ◽  
Yeast Saccharomyces Cerevisiae ◽  
Protein Coding ◽  
General Transcription Factor ◽  
Pol I ◽  
Pol Iii

The coordinated transcription of the genome is the fundamental mechanism in molecular biology. Transcription in eukaryotes is carried out by three main RNA polymerases: Pol I, II, and III. One basic problem is how a decrease in tRNA levels, by downregulating Pol III efficiency, influences the expression pattern of protein-coding genes. The purpose of this study was to determine the mRNA levels in the yeast mutant rpc128-1007 and its overdose suppressors, RBS1 and PRT1. The rpc128-1007 mutant prevents assembly of the Pol III complex and functionally mimics similar mutations in human Pol III, which cause hypomyelinating leukodystrophies. We applied RNAseq followed by the hierarchical clustering of our complete RNA-seq transcriptome and functional analysis of genes from the clusters. mRNA upregulation in rpc128-1007 cells was generally stronger than downregulation. The observed induction of mRNA expression was mostly indirect and resulted from the derepression of general transcription factor Gcn4, differently modulated by suppressor genes. rpc128-1007 mutation, regardless of the presence of suppressors, also resulted in a weak increase in the expression of ribosome biogenesis genes. mRNA genes that were downregulated by the reduction of Pol III assembly comprise the proteasome complex. In summary, our results provide the regulatory links affected by Pol III assembly that contribute differently to cellular fitness.

A common octamer motif binding protein is involved in the transcription of U6 snRNA by RNA polymerase III and U2 snRNA by RNA polymerase II

Cell ◽  
1987 ◽  
Vol 51 (1) ◽  
pp. 71-79 ◽  
Author(s):  
Philippe Carbon ◽  
Sylvie Murgo ◽  
Jean-Pierre Ebel ◽  
Alain Krol ◽  
Graham Tebb ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Binding Protein ◽  
Rna Polymerase Iii ◽  
U2 Snrna ◽  
U6 Snrna ◽  
Polymerase Iii ◽  
Octamer Motif

scholarly journals Parvovirus Expresses a Small Noncoding RNA That Plays an Essential Role in Virus Replication

2017 ◽  
Vol 91 (8) ◽  
Author(s):  
Zekun Wang ◽  
Weiran Shen ◽  
Fang Cheng ◽  
Xuefeng Deng ◽  
John F. Engelhardt ◽  
...  
Keyword(s):  
Dna Replication ◽  
Noncoding Rna ◽  
Rna Polymerase Iii ◽  
Human Bocavirus ◽  
Nonstructural Proteins ◽  
Viral Dna ◽  
Content Type ◽  
Small Noncoding Rna ◽  
Pol Iii ◽  
Tract Infections

ABSTRACT Human bocavirus 1 (HBoV1) belongs to the species Primate bocaparvovirus of the genus Bocaparvovirus of the Parvoviridae family. HBoV1 causes acute respiratory tract infections in young children and has a selective tropism for the apical surface of well-differentiated human airway epithelia (HAE). In this study, we identified an additional HBoV1 gene, bocavirus-transcribed small noncoding RNA (BocaSR), within the 3′ noncoding region (nucleotides [nt] 5199 to 5338) of the viral genome of positive sense. BocaSR is transcribed by RNA polymerase III (Pol III) from an intragenic promoter at levels similar to that of the capsid protein-coding mRNA and is essential for replication of the viral DNA in both transfected HEK293 and infected HAE cells. Mechanistically, we showed that BocaSR regulates the expression of HBoV1-encoded nonstructural proteins NS1, NS2, NS3, and NP1 but not NS4. BocaSR is similar to the adenovirus-associated type I (VAI) RNA in terms of both nucleotide sequence and secondary structure but differs from it in that its regulation of viral protein expression is independent of RNA-activated protein kinase (PKR) regulation. Notably, BocaSR accumulates in the viral DNA replication centers within the nucleus and likely plays a direct role in replication of the viral DNA. Our findings reveal BocaSR to be a novel viral noncoding RNA that coordinates the expression of viral proteins and regulates replication of viral DNA within the nucleus. Thus, BocaSR may be a target for antiviral therapies for HBoV and may also have utility in the production of recombinant HBoV vectors. IMPORTANCE Human bocavirus 1 (HBoV1) is pathogenic to humans, causing acute respiratory tract infections in young children. In this study, we identified a novel HBoV1 gene that lies in the 3′ noncoding region of the viral positive-sense genome and is transcribed by RNA polymerase III into a noncoding RNA of 140 nt. This bocavirus-transcribed small RNA (BocaSR) diverges from both adenovirus-associated (VA) RNAs and Epstein-Barr virus-encoded small RNAs (EBERs) with respect to RNA sequence, representing a third species of this kind of Pol III-dependent viral noncoding RNA and the first noncoding RNA identified in autonomous parvoviruses. Unlike the VA RNAs, BocaSR localizes to the viral DNA replication centers of the nucleus and is essential for expression of viral nonstructural proteins independent of RNA-activated protein kinase R and replication of HBoV1 genomes. The identification of BocaSR and its role in virus DNA replication reveals potential avenues for developing antiviral therapies.

scholarly journals Involvement of RNA Polymerase III in Immune Responses

2015 ◽  
Vol 35 (10) ◽  
pp. 1848-1859 ◽  
Author(s):  
Damian Graczyk ◽  
Robert J. White ◽  
Kevin M. Ryan
Keyword(s):  
Transcription Factor ◽  
Immune Responses ◽  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Trna Genes ◽  
Malignant Cells ◽  
Polymerase Iii ◽  
Mouse Macrophages ◽  
Tightly Coupled ◽  
Pol Iii

Inflammation in the tumor microenvironment has many tumor-promoting effects. In particular, tumor-associated macrophages (TAMs) produce many cytokines which can support tumor growth by promoting survival of malignant cells, angiogenesis, and metastasis. Enhanced cytokine production by TAMs is tightly coupled with protein synthesis. In turn, translation of proteins depends on tRNAs, short abundant transcripts that are made by RNA polymerase III (Pol III). Here, we connect these facts by showing that stimulation of mouse macrophages with lipopolysaccharides (LPS) from the bacterial cell wall causes transcriptional upregulation of tRNA genes. The transcription factor NF-κB is a key transcription factor mediating inflammatory signals, and we report that LPS treatment causes an increased association of the NF-κB subunit p65 with tRNA genes. In addition, we show that p65 can directly associate with the Pol III transcription factor TFIIIB and that overexpression of p65 induces Pol III-dependent transcription. As a consequence of these effects, we show that inhibition of Pol III activity in macrophages restrains cytokine secretion and suppresses phagocytosis, two key functional characteristics of these cells. These findings therefore identify a radical new function for Pol III in the regulation of macrophage function which may be important for the immune responses associated with both normal and malignant cells.

scholarly journals In vitro analysis of elongation and termination by mutant RNA polymerases with altered termination behavior.

1996 ◽  
Vol 16 (11) ◽  
pp. 6468-6476 ◽  
Author(s):  
S A Shaaban ◽  
E V Bobkova ◽  
D M Chudzik ◽  
B D Hall
Keyword(s):  
Rna Polymerase Iii ◽  
Multiple Roles ◽  
Wild Type ◽  
Protein Motifs ◽  
Study Support ◽  
Vitro Analysis ◽  
Pol Iii ◽  
In Vitro Analysis

We have studied the in vitro elongation and termination properties of several yeast RNA polymerase III (pol III) mutant enzymes that have altered in vivo termination behavior (S. A. Shaaban, B. M. Krupp, and B. D. Hall, Mol. Cell. Biol. 15:1467-1478, 1995). The pattern of completed-transcript release was also characterized for three of the mutant enzymes. The mutations studied occupy amino acid regions 300 to 325, 455 to 521, and 1061 to 1082 of the RET1 protein (P. James, S. Whelen, and B. D. Hall, J. Biol. Chem. 266:5616-5624, 1991), the second largest subunit of yeast RNA pol III. In general, mutant enzymes which have increased termination require a longer time to traverse a template gene than does wild-type pol III; the converse holds true for most decreased-termination mutants. One increased-termination mutant (K310T I324K) was faster and two reduced termination mutants (K512N and T455I E478K) were slower than the wild-type enzyme. In most cases, these changes in overall elongation kinetics can be accounted for by a correspondingly longer or shorter dwell time at pause sites within the SUP4 tRNA(Tyr) gene. Of the three mutants analyzed for RNA release, one (T455I) was similar to the wild type while the two others (T455I E478K and E478K) bound the completed SUP4 pre-tRNA more avidly. The results of this study support the view that termination is a multistep pathway in which several different regions of the RET1 protein are actively involved. Region 300 to 325 likely affects a step involved in RNA release, while the Rif homology region, amino acids 455 to 521, interacts with the nascent RNA 3' end. The dual effects of several mutations on both elongation kinetics and RNA release suggest that the protein motifs affected by them have multiple roles in the steps leading to transcription termination.

Eukaryotic transcription termination factor La mediates transcript release and facilitates reinitiation by RNA polymerase III

1994 ◽  
Vol 14 (3) ◽  
pp. 2147-2158
Author(s):  
R J Maraia ◽  
D J Kenan ◽  
J D Keene
Keyword(s):  
Rna Polymerase ◽  
Rna Binding ◽  
Transcription Termination ◽  
Rna Polymerase Iii ◽  
Class Iii ◽  
Ample Evidence ◽  
Polymerase Iii ◽  
Transcription Complexes ◽  
Pol Iii

Ample evidence indicates that Alu family interspersed elements retrotranspose via primary transcripts synthesized by RNA polymerase III (pol III) and that this transposition sometimes results in genetic disorders in humans. However, Alu primary transcripts can be processed posttranscriptionally, diverting them away from the transposition pathway. The pol III termination signal of a well-characterized murine B1 (Alu-equivalent) element inhibits RNA 3' processing, thereby stabilizing the putative transposition intermediary. We used an immobilized template-based assay to examine transcription termination by VA1, 7SL, and Alu class III templates and the role of transcript release in the pol III terminator-dependent inhibition of processing of B1-Alu transcripts. We found that the RNA-binding protein La confers this terminator-dependent 3' processing inhibition on transcripts released from the B1-Alu template. Using pure recombinant La protein and affinity-purified transcription complexes, we also demonstrate that La facilitates multiple rounds of transcription reinitiation by pol III. These results illustrate an important role for La in RNA production by demonstrating its ability to clear the termination sites of class III templates, thereby promoting efficient use of transcription complexes by pol III. The role of La as a potential regulatory factor in transcript maturation and how this might apply to Alu interspersed elements is discussed.

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