Cytokinesis Flashcards

Question

Actin network is essential for ordered inheritance

Answer 1

- Transient linkage of Mito to cortical actin cables via myosin are required for equal segregation of Mito mass - Comet tails of actin are importuned for mixing of Mito during mitosis, thus probably allowing equal distribution of different "types" of Mito to daughter cells.

Answer 2

G1 -> G1-S (elongated) -> G2 and M (fragmented) -> M (individual) -> G1 - Machinery for maintenance of morphology (balance between fission and fusion) and distribution into daughter cells is linked. - Balance between fusion and fission is important for maintenance of mitochondria morphology

Answer 3

Dmn1 - dynamin-related GTPase; the colocalisation of fission site, ERMES, mtDNA and ER-Mito contact seems to be the typical situation Recruitment of Dynamic 2 -> final fission

Answer 4

- Bacteria: FtsZ GTPase (homologous to tubulin) assembles to a spiral ring and induces septum formation - red Algea: FtsZ assembles in the Mito inducing reconstriction and recruitment of dynamic-like Dnm1 GTPase which finally results in contriction and division - yeast: ER contact sites induce preconstriction and recruitment of Dnm1

Answer 5

- Fzo1-Mgm1-Ugo1 complex required for fusion. - Fzo1: dynamic related transmembrane GTPase of the outer membrane - Mgm1; dynamic related GTPase of the inter membrane space - Ugo1: outer membrane protein

Answer 6

- Prefusion: Monomer -> GTP - Tethering: Extended dimer -> GTP - Docking: Closed dimer -> GDP - Hemifusion: Bent dimer -> GDP - Postfusion: Monomer -> GDP -> GTP

Answer 7

- manifestation of mitochondrial Enzephalomypathies depends on load of pathogenic mtDNA in the tissues (onset between 60 ... 90 %) - mixing of mitos via fusion allows in heteroplastic cells the delivery of non-mutated products to mitos with mutated genomes - mitochondrial DNA is packed in nucleoids which contain several DNA copies; a given mitochondrion has several nucleoids -> mixing of mitos mixes nucleoids -> mixing of mutated and wt DNA -> homogenization of mitos -> progression of homoplasmy (cells/tissues with only mutated mitos) is damped - fission is asymmetric, separating more healthy parts of mitochondria from others having a higher load of damaged molecules, which later can be eliminated by autophagy - Fusion of badly damaged mitos with very low inner membrane potential is blocked by inactivation of the dynamic-related GTPase OPA! (inner membrane), MNF1 and 2 (outer membrane) and other molecules -> some rare neurological diseases are caused by defects in mito fission or fusion

Answer 8

- observed in different cell types mainly with mitochondria with low mobility - product of incomplete fission or also of an active process with mobile protrutions - proposed function: exchange soluble and membrane proteins

Answer 9

- rare (?) event - at least two versions -> DRP1-dependent vesicles -> DRP1-independent vesicles destined for peroxisomes - precise function unknown

Answer 10

- best investigated example of nuclear segregation in eukaryotes with closed mitosis - inheritance of nucleus and ER are tightly coupled

Answer 11

- beginning with early anaphase, the bud neck blocks diffusion of OM protein complexes between mother and daughter cell; block has to be released actively in order to allow distribution of NPC into daughter cell which results in a relative enrichment of old NPC in the mother cell - NPCs with non-functional Nsp1p subcomplexes are retained in the mother cell - molecules thought to be attached to NPC, like CEN-free plasmids, are retained in the mother nucleus

Answer 12

- budding yeasts have no Golgi-stacks - only cis-Golgi is close to ER-exit sites - partially de novo construction of early Golgi (Pichea pastoris) (late Golgi elements come from existing Golgi structures) - in yeast late Golgi elements are translocated via Myso2p and actin into the bud-tip

Answer 13

- vacuoles in yeast are dynamic, 1-5 per cell - hamotypic fission and fusion essential for vacuolar inheritance - vacuole inheritance initiates early in the cell cycle and ends in G2, just prior to nuclear migration 1) tubulation 2) vesiculation/prevent fusion 3) migration into bud 4) fusion

Answer 14

All SNAREs needed are in both membranes! Activation of SNAREs by dissembling of the cis-complexes occurs at both membranes! -> Tethering -> Fusion of SNAREs to trans-complexes

Answer 15

Although it is presently believed that are least in yeasts peroxisomes may form de novo, also yeast cells have a mechanism to deliver peroxisomes to the daughter cell

Answer 16

- during cell growth the part of mitochondria near the neck region extends into the bud tip and growth their while the rest of the mother mitochondrial network with most of the old nucleotide is retained in the mother cell

Answer 17

- Meiosis II/Prospore Membrane growth + Prospore Membrane Closure: Formation of Prospore-membrane by vesicles coming from the trans Golgi - Spore Wall Assembly/ Asocial Maturation: Formation of the spore cell wall

Answer 18

- duplication and inheritance by precise distribution (e.g centrosome with centrioles) - unequal distribution of large cytosolic or PM complexes (e.g. protein aggregates in yeast and cells of higher eukaryotes) -> one possibility is the linkage to actin cables via motors -> another is the confinement of aggregates e.g. due to binding to ER or parts of mitochondria that are retained in the mother cell

Answer 19

Egg brings bulk of cytosol and organelles

Answer 20

1. Prefusion a) Acquiring of fusion competence (=differentiation) b) Chemotaxis (whole cell movement or formation of filipodia or similar protrusions) c) cell cell recognition ("tethering"; "docking"; may include protein protein interaction, lipid remodeling, protein-lipid interaction) leading to cell-cell adhesion 2. Fusion ( = needs fusogen) - formation of a (most transiently existing) fusion pore; - mixing of the luminal content; - usually also mixing of the membranes

Answer 21

- actual fusion process not well characterized - HAP1-GCS1 involved in gamete fusion of viridaeplantae and different protists, most likely acting as a genuine fusogene (homology to Type II fusogenes of virus) - protein factors involved in Mammalia are CD9 (on egg), Izumo1 (on sperm), ADAM1 and ADAM2; unclear whether these factors are directly involved fusogens - other, unrelated factors (egg and spe proteins) are needed in C. elegans; only spe45 is homolog to Izumo1

Answer 22

1) Formation in vivo normal (e.g. formation of muscle cells, osteoclasts or placenta) 2) Spontaneous formation in vitro and in artificial system in vivo shown; normal formation in vivo under debate (observed e.g. in Ruminantia) 3) Formation of synkaryons in vivo so far only in artificial systems observed; viral proteins may act as fusogens (e.g. HERV-W = syncytin)

Answer 23

Several syncytial tissues; induction by homotypic interaction of one fusogen (EFF-1) that is aided by a regulating factor and that is specific for this clade!!

Answer 24

- myoblast differentiation - acquisition of fusion competence - recognition and adhesion of cells by receptors - signaling from receptor to actin -> actin remodeling; formation of podosomes (invadosomes); involvement of endocytosis - transient exposure of PtdSer on outer leaflet, binding of annexing - activation/transport to the PM - fusion by step-wise activity of two proteins - myomaker induces hemifusion while myoperer performs the step from hemifusion to fusion (formation of the fusion pore)

Answer 25

- fusion mainly (exclusively?) by syncytin, a fusogen decending from retroviruses, binding to its cognate membrane receptor

Answer 26

- originate from mononucleated macrophages on demand - recognition and adherence by a series of receptor interactions with first long and later short extracellular regions - signaling to the nucleus - rearrangement of cytoskeleton, cell-cell and cell-substrate adhesion (e.g. via E-cadhrine and integrins) - signaling from receptor to actin -> actin remodeling; formation of podosomes (invadosomes); involvement of endocytosis - exposure of PtdSer at the outer leaflet - fusogen still unknown (2017) although first indications for a fusion by Syncytin-1

Answer 27

- cell-cell communication by spontaneous intercellular transfer of cellular components (ICT) in vitro - transfer of PM membrane proteins and cytoplasmic particles below 50 kDA between somatic cells - transfer of larger particles (2000 kDa) observed between stem cells and somatic cells!

Answer 28

- eukaryotic cells have a constant nucleoplasm to protoplasm ratio - continuous increase of cell size during cell cycle is accompanied by a continuous increase of the nuclear size - in artificial multinucleated cells bearing nuclei of different size, the size of a particular nucleus is proportional to the volume of the surrounding protoplasm -> the energized behaves like a cell

Answer 29

Some cells have nuclei of non-ovoid shape, e.g. polymorphonuclear leukocytes. The nuclear shape of neutrophils, which belong to this cell type, is controlled by lamins, laminB receptor and the cytoskeleton

Answer 30

The ER may adopt to forms - tubules or sheets

Answer 31

ER tubules are shaped by two protein families: - Reticulons - DP1/REEP 5 (mammals); Yop1p (S. cerevisiae)

Answer 32

Hydrophobic hairpins could form a "wedge" + Homo-, hetero-oligomerize into arc shapes -> would induce the same curvature in tubules and at the edges of sheets

Answer 33

- proteins involved are either Atlastins (e.g. mammals) or Sey1p (yeast) - while Atlastins seems to be essential, Sey1p in yeast is not essential -> involvement of ER-SNAREs? - process need GTP hydrolysis

Answer 34

- dynamic-like GTPases; anchored in ER membrane via C-terminal TM segments (similar topology as the dynamic-like mitofusins) - interact with Reticules and DP1 - interact in trans (resulting in a tethering OR fusion of ER domains) and in cis; the latter may be important for the formation of the three-way junctions in the network - can induce tubules and thus form networks in vitro alone

Answer 35

Three-way junctions are small sheets with concave edges - protein involved in junction formation is Lunaparke supported by atlastin - overexpression of reticulon or Yop1p can substitute the loss of lunapark

Answer 36

- the curvature induction at the edges by reticulons is one possible mechanism - in mammalian cells are additional factors like Climp63 kinetin or p180

Answer 37

- overexpression of tubule-formers leeds to less sheets; k.o. of junction-formers results in sheets but can be substituted by overexpression of tubule-formers -> balance between the different components is crucial for the morphology the ER of a cell - proteins or complexes like membrane-bound polysomes adapted to a special ER-form may in turn also have some influence in stabilizing/inducing this form - strong induction of ER-tubules blocks the nuclear envelope reformation after open mitosis other players under discussion: - proteins of the tip attachment complex (TAC) which couple ER to growing microtubule tips (involved in stretching the ER network over the MT-system) - spastins (cleavage of tubules?) - Rab10 (involved in tubule tip growth) - Rab18 (regulates balance between tubules and sheets)

Answer 38

Stacked ER sheets form a continuous membrane system in which the sheets are connected by twisted membrane surfaces with helical edges of left- or right-handedness. A theoretical model explains the experimental observations (number and shape of connections, nearly equal abundance of left and right-handedness, distance between stacks) especially also their dependence on length and curvature of the edges and indicates that the structure corresponds to a minimum of elastic energy of sheet edges and surfaces. -> ER sheets are connected by membranes -> Golgi sheets by protein bridges

Answer 39

- diffusion barriers formed by septins may limit diffusion of ER membrane proteins forming polarized ER - barrier examples are the neck of budding yeast or dendritic branch points

Answer 40

- size and dynamic of the network - size and number of "single" mitochondria - number and appearance of cristae

Answer 41

- cristae junction define the border between two subcompartments of the inner mitochondrial membrane - cristae junction probably define also a border between two subcompartements of the intramembrane space - the difference in protein enrichment of particular proteins in the two subcompartments is dynamic

Answer 42

- shaping of cristae is performed by balanced activity of MIC60, MIC10, cardiolipin, Phosphatidylethanolamin, FoF1ATPase, Opa1 and MICU - In MICOS (earlier also called MINOS, MitOS a.o.) mutant mitochondria, most crista junctions are lost and large internal membrane stacks, resembling a labyrinth, are observed. - MICOS is enriched in the vicinity of crista junctions. It interacts with protein translocates of the outer membrane (TOM, SAM), the channel-forming protein porin (VDAC), the outer-membrane fusion component Ugo1 and Mia40.

Answer 43

- cristae have higher delta psi compared to their adjoining inner mitochondrial membranes - cristae are electrically insulated, allowing individual cristae within any given mitochondria to have different membrane potentials - cristae can remain polarized despite depolarization of neighboring ones - disruption of crista junctions impairs the electrical insulation of cristae, equilibrating their delta psi with those of inner mitochondrial membranes -> Cristae undergo continuous cycles of membrane remodeling in a MICOS-dependent manner

Answer 44

1) Biogenesis of beta-barrel precursors 2) Biogenesis of Tom22 and Tom40 3) Shuttling between SAM and ERMES 4) Mitochondrial morphology and lipid homeostasis

Answer 45

- Mitochondrial-derived compartments (MDCs) are micron-sized organelle subdomains that form in response to elevated amino acid levels - are stable associated with mitochondrial network and in contact with ER via ERMES - have specific shape and protein pattern and lack mtDNA - support the catabolism of AA via Ehrlich-pathway to so-called Fusel Alkohol

Answer 46

- Distribution and positioning is aided by the cytoskeletal network - appearance of this network is determined by inner factors and by the extracellular surrounding (e.g. contact sites with neighboring cells or the ECM) - moreover, distinct contact sites are formed between organelles

Answer 47

degradation of mature RNA is largely exonucleolytic - protection of ends by caps, strong tertiary structures and/or binding proteins - removal of these structures or endonucleolytic cleavage/ strand break activates decay

Answer 48

- part of enzymes involved in biosynthesis of -> chromosomes (e.g. telomerase) -> ribosomes (e.g. snoRNA, MRP RNA) -> tRNA (H1 RNA) -> spliceosomes (sca RNA) -> mRNA (spliceosomal RNA) -> proteins (rRNA, tRNA, 7SL RNA) - transfer of information (mRNA) - regulation of gene expression

Answer 49

1) Chromatin accessibility 2) Activator/repressor binding and function 3) Transcription initiation 4) Transcription elongation 5) RNA processing and modification 6) RNA stability 7) mRNA translation

Answer 50

RNA acting by base pairing - cis-encoded - trans-encoded RNA modifying protein activity -acting at transcription - acting at translation RNA modifying activity of regulatory RNA

Answer 51

- are encoded at the same genetic location; but on the opposite strand to the RNAs they act upon - source: -> bacterial plasmids: mainly to regulate the transcription, stability, or translation of mRNA encoding proteins critical for replication or stable plasmid inheritance -> transposons -> phages -> chromosomal genes, but only few examples with identified functions (e.g. some forms of siRNA)

Answer 52

pT181 - negative feed-back loop controlling copy number - plasmid copy number (up-regulation) -> RNAI (85 nt) (up-regulation) and RNAII (150 nt) antisense RNAs -> RNAs base pair with and stabilize a structure associated with transcription termination upstream of the coding sequence for replication regulator RepC -> less RepC -> no replication -> plasmid copy numbers decrease (down regulation) -> RNAI and RNAII levels decrease (downregulation) -> RepC levels (upregulation) -> renewed replication R1 - example for plasmid addiction system - plasmid has a hok-sok gene that codes for two RNAs -> hok (host killing) -> a mRNA coding for a small toxic protein -> sok (suppressor of killer) -> a RNA repressing hok mRNA translation When the plasmid is lost, the differential stability of the sok and hok RNAs determines that the sok RNA levels decrease faster than the hok mRNA levels -> Hok protein expression -> damage of the bacterial membrane -> cell death

Answer 53

- are encoded at a chromosomal location distinct from the RNAs they act upon Examples: - Hfq-binding RNAs from bacteria -> >10 examples from E.coli -> bind to a common protein, Hfq -> this complex destabilizes mRNAs and hence either represses or activates translation -> involved in regulation of physiological switch in response to carbon sources, iron supply or quorum sensing - eukaryotic miRNAs - eukaryotic snoRNAs

Answer 54

- transcription modulation, examples -> bacterial 6S RNA binding + inhibiting promoter-specific sigma70-polymerase -> mammalian 7SK RNA binding + inhibiting transcription elongation factor P-TEFb - mouse B2 RNA binding + inhibiting promoter specific RNAse II after heat-shock - Modulation of mRNA stability and translation -> bacterial CRSB/RSMY family of RNAs binding + sequestering CsrA + RsmA proteins which regulate decay of mRNAs -> human dendritic B1 RNA binding and activating FMRP interaction with target mRNAs

Answer 55

Xist performs multiple roles during X chromosome inactivation (XCI) by recruiting various regulatory complexes to enact and maintain chromosome-wide transcriptional silencing.

Answer 56

- 50 % of primary Pol I transcripts are degraded in the nucleus - 95 % of primary Pol II transcripts are degraded in the nucleus -> Major site of maturation and quality control

Answer 57

- quality control circuits for RNA often can be viewed as kinetic competitions between the rate of the reaction controlled by this RNA and a quality-control event targeting the RNA for degradation -> proper RNP promotes normal function: decreases time for quality control of normal RNA -> Aberrant RNP promotes quality control: targets aberrant RNA for quality control -> Aberrant RNP inhibits normal function: increases time for quality control of aberrant RNA

Answer 58

The majority of ribosomal proteins (r-proteins) need to be transported from the cytoplasm to their assembly sites within the nucleus. The assembly pathways leading to the formation of mature 40S and 60 S subunits originate from a common pre-rRNA (35S pre-rRNA) that is transcribed by RNA polymerase I. Distinct assembly modules, biogenesis factors, and r-proteins associate cotranscriptionally with the nascent pre-rRNA to form a 90S preribosome. Dismantling of the 90S pre ribosome and pre-rRNA cleavages liberate the 50-external transcribed spacer (ETS) complex, whose components are recycled for further assembly rounds, and the first pre-40S particle. Export factors mediate the transport of pre-40S ribosome to the cytoplasm where they undergo further maturation steps, including beak formation. Final 40S maturation occurs within 80S-like ribosomes and couples pre-rRNA cleavage with a quality control step. The first pre-60S particle, whose components begin to associate with the nascent pre-rRNA, is likely to be formed after internal transcribed spacer 1 (ITS1) cleavage and termination of transcription. Then, the preformed 5S ribonucleoprotein particle (RNP) associates with early-pre-60S particles, which already contain the ITS2-associated biogenesis factors. Subsequently, ITS2 processing is initiated and the first Rea1-dependent remodeling step occurs. The Nog2-purified pre-60S particle exhibits the prominent "foot" structure and the 5S RNP in its premature position. The second Rea1-dependent remodeling step coincides with rotation of the 5S RNP into its mature position and occurs in concert with the release of the GTPase Nog2, which is a prerequisite for recruitment of the export adaptor Nmd3 and translocation through the nuclear pore complex. In the cytoplasm final maturation and quality control steps yield mature 60S subunits.

Answer 59

- about 33 positions in E.coli with 95 % in functional important regions - about 108 position in yeast with 60 % in functional important regions - about 206 in human Main types: a) 2'-O-methylation (Nm) (in human 50 % of mod. bas.) b) isomerization of uridine to pseudouridine (psi) (in human 45 % of mod. bas.) c) base methylation at various positions; this includes psi A few are acetylated or otherwise modified at such positions. Enzymes: - in E.coli only "protein-only" enzymes - in eukaryotes (exclusive) involvement of RNA containing enzymes in Psi and Nm modification

Answer 60

- Box C/D snoRNAs guide 2'O methylation - Box H/ACA snoRNAs guide pseudouridylation

Answer 61

- in the nucleus: several pathways, common feature is adenylation and 3' to 5' decay by the exosome or Rrp6p and/or 5' to 3' decay by Rat1p - in the cytoplasm: nonfunctional rRNA decay (NRD) - 18S NRD, 25S NRD

Answer 62

- in the nucleus: several pathways, common feature is adenylation and 3' to 5' decay by the exosome or Rrp6p

Answer 63

- divergent structures in eukaryotes - length of the RNA: 150 nt in ciliates, 400-600 nt in vertebrates ca 1300 nt in yeast - RNA polymerase III transcribes it in ciliates; RNA polymerase II transcribes the yeast and vertebrate telomerase RNA - biogenesis involves nucleolus and Cajal bodies (human form contains scaRNA-motif)

Answer 64

- data mainly based on yeast - pre-tRNA transcription -> first modifications -> binding of La protein to 3' end -> processing of the 5' leader by RNAse P (inside or outside the nucleolus) -> processing of 3' leader by endo- or exonuclease -> modifications -> addition of CCA to 3' end -> splicing of intron (if the particular pre-tRNA has one); may also proceed processing of 5' and 3' ends! - in yeast the splicing occurs in the cytoplasm in the vicinity of mitochondria

Answer 65

IN THE NUCLEUS tRNA modification involved in quality control -> simultaneous lack of several modifications in yeast leads to rapid TRAMP-dependent adenylation and 3'-5' decay by the exosome IN THE CYTOPLASM degradation: -> rapid tRNA decay (RTD) mature tRNA can in part be regenerated (e.g. re-adding of CCA) -> very stable - back transport of tRNA in yeast and vertebrates (function: maturation/quality control/repair/degradation/down regulation of translation?)

Answer 66

Aberrant non-coding RNA is polyadenylated which marks them for degradation by the exosome

Answer 67

- presently > 200 different forms known (about 100 in worm, > 150 in human, > 20 in plants) with about 1/3 being conserved between phyla of Metazoa and nearly all being conserved within classes of Metazoa - most miRNA come from own transcriptional units (sometimes several from one primary transcript), but a significant number comes from introns of functionally related protein transcripts

Answer 68

- RNAs defined by their binding to the piwi-protein family - present in all Metazoa (exception Trichoplax) - transcribed from a few hot-spots in the genome - proposed function: down regulation of transcripts from transposons thus controlling their spreading in the genome

Answer 69

- siRNAs often derive from mRNAs (e.g. product of pseudogenes), viruses or transposons - siRNAs are processed from long bimolecular RNA duplexes or extended hairpins - while a single miRNA:miRNA-duplex is generated from each miRNA a multitude of siRNA duplexer are generated from each siRNA precursor, leading to many different siRNAs accumulating from both strands of this extended dsRNA - many siRNA are exogenous; endogenous siRNA sequence are rarely conserved

Answer 70

- Extensive complementarity in coding region or UTR: mRNA cleavage (miRNA or siRNA) - Short complementary segments in 3'-UTR: translational repression (miRNA or siRNA) - Interaction with DNA: transcriptional silencing (rasiRNA)

Answer 71

- mature form stems from intronless transcript and has size comparable to mRNAs; >16000 loci in human genome - only 3 % which may have mainly cell-type specific functions have been studied in detail (e.g. XIST which is crucial for random X-chromosome inactivation) - mostly unspoiled and PolyA-free and hence short-lived in the cytoplasm due to exosome activity - may regulate in cis and in trans - regulate in the nucleus -> transcription (acting on proteins like transcription factors or modulators of chromatin) by different mechanisms (recruiting factors, modulating activity of factors, displacing factors) -> mRNA splicing and editing (NATs) - regulate in the cytoplasm -> translation activity -> mRNA half-life by directing nucleases to the mRNA or by inhibiting miRNA

Answer 72

The mRNA-pool is an important actuator for the regulation of protein levels end hence gene expression. - stable mRNA (e.g. beta-globing or maternally stored mRNA in oocytes ....) may last several cell cycles - instable mRNA (e.g. cell-cycle dependent histones, cytokines ...); in E-coli half life 30 s to 20 min; in mammalian cells half life is usually longer but sometimes as short as 1/100 of generation time - most mRNAs are stabilized in mitosis or in the presence of translation inhibitors - other factors regulating mRNA stability are development/differentiation (e.g. global mRNAs, cytokines, photo-oncogenes), physiological states or stress - mRNA stability may get deregulated (carcinoma, Alzheimer ...)

Answer 73

Causes for aberrant mRNA: - mutations in the gene - errors during transcription or processing - fuzziness or alternative splicing sites - damage Quality checkpoints exist: - (after transcription ???) - after splicing and before export - during each round of translation termination

Answer 74

- deadenylation dependent pathway (used by the bulk of mRNA) - deadenylation independent pathway (rare) - endonuclease mediated pathway (highly regulated or highly specific)

Answer 75

1) Poly (A)-binding protein and cap-binding protein (eIF4E) associate with the mRNA 2) Interaction of Pab1 with translation initiation factor eIF4G promotes formation of the "closed loop", initiates translation and antagonizes decapping 3) Translation termination by an 80S ribosome is promoted by recognition of the UAG codon by eRF1 and stimulated by interactions between eRF1 and eRF3, and between eRF3 and Pab1 4) The poly(A) tail of the mRNA is shortened by the 3'-to-5' exonuclease CCR4-NOT, a process that continuous through multiple rounds of translation. Poly(A) shortening minimizes the number of Pab1 molecules associated with the 3'-untranslated region ultimately disrupting the closed loop. 5) Loss of the poly(A) tail and Pab1 facilitates an mRNP rearrangement in which the dissociation of eIF4E from the 5'-cap and the binding of the Lsm1-7-Pat1-Dhh1 complex stimulates Dcp1-Dcp2-mediated decapping, followed by degradation by the 5'-to-3' exonuclease Xrn1. Alternatively, the transcript can become a substrate for the 3' to 5' exonucleolytic exosome.

Answer 76

Instability can occur due to; I. the presence of destabilizing elements or II. a regulating element, that effects stabilizing elements (e.g. PolyA) or the access to destabilizing elements or III. a lack of usually present stabilizing elements (e.g. PolyA) combined with the presence of other special regulated stabilizing elements Localization: - 3' UTR elements or - Elements in coding region or - Elements in 5' UTR Some elements species can be present in different positions, depending on the type of mRNA.

Answer 77

other elements in 3' UTR: - human alpha-globulin - frog albumin elements in 5' UTR: - JNK response element (JRE) in IL2 mRNA (binding factors are nucleoline and YB-1) - 68 nt Element in KC mRNA

Answer 78

- typical sequence tries of overlapping AUUUA pentamers which in mammalians can be grouped in classes - ARE mediated decay regulates 5-8 % of cellular mRNA - usually in 3' UTR - in short-lived mRNAs like cytokines or proto-oncogenes - regulating factors: -> TTP, AUF1 complex (which interacts with HSP70) and others accelerate destabilization -> ELAV-homologs like HuR stabilizes -> GAPDH and other bind ARE with unclear effect - degradation is performed by exosomes

Answer 79

- IRE are stem loops which bind iron response proteins (IRP) in the absence of iron - IRE are found in the 3' UTR of transferring receptor (TfR), and in the 5' UTR of mitochondrial aconitase, erythroid delta-aminolevulinic acid synthase and ferritin H and L chain and in other mRNAs of Metazoa or bacteria coding for proteins that function in iron uptake, storage, and export, in heme synthesis or the tricarbocylic acid cycle/ATP production

Answer 80

- in the presence of iron IRP1 binding activity gets inactivated; IRP2 gets degraded by proteasome

Answer 81

Iron Response Proteins (IRP) binding to IRE blocks access of 43S initiation complex to its binding site

Answer 82

some examples: - CRD1 (purine-rich) and CRD2 element of c-fos -> interacting with complex that has AUF1 homolog - first 13 nt of beta-tubulin - CRD-like element in c-myc

Answer 83

- Histone mRNAs lack poly(A) tails - cell-cycle dependent decay is mediated by a 3' terminal stem-loop motif (6 bp stem and a 4 base loop) - regulation via stem-loop binding protein

Answer 84

- degradation of mRNA lacking Poly(A) by nuclear form of exosome - degradation of mRNA lacking termination codons by exosome in the cytoplasm (no story decay - NSD) - transcript with aberrant termination behavior by nonsense-mediated decay (NMD) pathway (=mRNA surveillance senso stricto) - degradation of mRNA with stalled ribosomes (e.g. due to a strong secondary RNA-structure) (no-go decay - NGD) - if these defective mRNA result in aberrant nascent proteins, they are degraded via the ubiquitin-proteasome system using specific E2/E3

Answer 85

NMD is activated, when a termination codon is located outside of a permitted distance to a downstream marker thus being identified as "premature termination codon" (PTC). The machinery recruited is in all cases similar. Invertebrates: DSE = downstream sequence Element (e.g. Faux 3' UTR) Mammals: EJC = exon junction complex

Answer 86

NORMAL TERMINATION The ribosome approaches the stop codon engages the UAG in the A-site, binds the release factors Sup45 and Sup35, and releases the polypeptide. Normal termination is thought to be highly efficient because interactions between the poly(A)-binding protein Pab1 and Sup35 enhances the ability of Sup35 to stimulate Sup45. The effect of Pab1 depends on its proximity to the termination event. PREMATURE TERMINATION The ribosome approaches a premature UAG codon, engages the UAG in it's A-site, binds Sup45 and Sup35, but fails to release the polypeptide. The NMD factors Upf1, Nmd2 and Upf3 bind to the release factors, stimulating peptide hydrolysis and 60S ribosomal subunit dissociation. Association of NMD factors with the ribosome triggers recruitment of the Dcp1-Dcp2 decaying enzyme complex and promotes mRNA decapping. Premature termination is thought to be inefficient because the termination site lacks proximal Pab1 and/or other factors that are associated with a natural 3'-UTR.

Answer 87

- degradation of aberrant transcripts with PTC: -> point mutations -> due to false initiation in the 5' UTR -> due to false splicing - degradation of about 10 % of the normal transcriptome -> selenoprotein mRNA with unused selenocysteine UGA -> mRNAs characterized by upstream ORFs, introns within their 3' UTRs, or nonsense-containing transposon or retroviral sequences within their coding regions -> alternatively spliced mRNA (in human about 1/3 of all mRNA!); NMD is not perfect, therefore 5-25 % of the PTC containing mRNA survive -> way of regulation of the level of alternative spliced versions - gene regulation PTCs are found in 30 % of inherited genetic disorders; Nonsense mediated decay may here be positive but ineffective (degradation of mutated mRNA which would cause a dominant phenotype) or negative (degradation of a mutated mRNA which could give rise to a (partially) functional protein)

Answer 88

High concentration of SC35 activates splicing events within the 3' UTR of SC35 pre-mRNA. As a consequence, exon joining complex appear downstream of the normal termination codon (Norm Ter) that elicit non mediated-decay when translation terminates normally.

Answer 89

- translation beyond normal stop codon into 3' UTR due to anti termination mutations - probably rare and cell-type restricted

Answer 90

- processing bodies (P-bodies) - stress granules -> germ cell granules - neuronal granules belong to the class of membrane-less compartment-like structures which assemble and keep their identity due to liquid-liquid phase separation driven a.o. by low complexity domains in proteins Phase separation occurs when molecules can achieve the same low free energy by adopting two distinct types of configuration with disparate concentrations and extents of intermolecular contacts. The low free energy is achieved by distinct means in two phases: for example high entropy (due to low concentration) in the dispersed phase but strong favorable (i.e. negative) enthalpy (from intermolecular contacts) in the droplet phase.

Answer 91

- observed in opistokonta (yeast and mammalian cells) - does not require eIF2alpha phosphorylation - enzymes that are required for the general mRNA decay pathway, including a deadenylase (CCR4), a decaying enzyme complex, an exonuclease (XRN1), an Lsm1-7 heptamer that regulates various aspects to RNP assembly and components of the nonsense-mediated decay pathway (e.g. SMG5, SMG7 and UPF1) - no ribosomal subunits (?!) - contain in mammalian cells components of the RNA-induced silencing complex (e.g. argonaute and microRNA (miRNA)) - stress (e.g. mRNA decay impairment; 5' -> 3' decay overload) induce PB - polysome disassemble causes PB assemble in yeast (which shoes no SG) - site of miRNA-mediated translational silencing in mammalians - site of ARE-mediated decay PB may vary in their protein composition (and hence function) within one cell

Answer 92

- observed in plant cells and cells from Metazoa (mammalians), but not in yeast - induced (15-30 min) after stress - dissolved: Ub dependent (heat stress) or independent (other stresses) - degraded by autophagy in case of long stress duration - requires eIF2alpha phosphorylation - 48S preinitiation complexes are the core constituents - contain also PABP1, the p54/Rck helicase, the 5'-3' exonuclease XRN1, and many RNA-binding proteins - selectively exclude mRNAs encoding stress-induced HSPs - polysome disassemble causes SG assemble in mammalians - may act as intermediates between polysomes and PB(?) - may accumulate components of the nuclear-cytoplasmic transport; thus interfering with it

Answer 93

- 50 % of PB have contact to ER - number of PB and PB contact site depends on translational activity at the ER - contact sites define point of division of PB

Answer 94

Germ cell granules in primodial germ cells if insects (e.g. Drosophila melanogaster), C. elegant, X. leavis -> contain maternal mRNA required for germ cell specification and proteins that regulate mRNA translational initiation, translation control and mRNA decay -> promote germ cell development in the early embryo and establish the germ line for the next generation

Answer 95

- found in neurons - harbor translational silenced mRNAs that are transported to dendritic synapses, where they are released and translated in response to exogenous stimuli = highly specific cargo - contain mRNA, small and large ribosomal subunits, translation initiation factors (e.g. eIF4E and eIF2alpha), and RNA-binding proteins that regulate mRNA function (e.g. HuD, G3BP, Sam68, SYNCRIP, hnRNP A2, RNG105, FMRP and Staufen)

Answer 96

- PAN2-PAN3: PABP-dependent shortening of Poly(A)-tails to about 60-80 nt - CCR4-NOT: PABP-inhibited deadenylation - PARN: cap-dependent deadenylation many more enzymes in mammalian cells proposed (spatial-temporal regulation, substrate specificity?)

Answer 97

Xrn1p and Rat1p, need 5'-monophosphate

Answer 98

IRE1 putative endonuclease that targets actively translating mRNAs that are usually targeted to the endoplasmic reticulum (ER) as part of the unfolded-protein response Ago2 usually si/miRNA dependent activity RNase MRP in nucleolus, but in mitosis in P-body like TAM-bodies in S. cerevisiae; degrades the CLB2 mRNA, which encodes a B-type cyclin, at the end of mitosis

Answer 99

- cytoplasmic form and nucleoplasmic form with 10 shared subunits (among them a 3'->5' hydrolytic exonuclease; several 3'->5' phosphorylase related exonuclase like subunits (lacking the active site); RNA helicases) - homologue in archea - nuclear exosome: -> 3' processing of 5.8S rRNA, snoRNA and maturation of snRNA = Trimming!! -> degradation of pre-rRNA spacer regions -> responsible for the surveillance and degradation of aberrant and excessive nuclear precursors of RNA including pre-mRNAs, pre-tRNAs and pre-rRNAs -> degradation of cryptic transcripts -> also implicated in regulated 3' degradation of some mRNAs to control their expression - cytoplasmic exosome -> turnover of mRNA -> degradation of aberrant mRNA (NMD, non-stop decay, no-go decay (stalled ribosomes)) -> degradation of short-lived mRNA (with ARE)

Answer 100

The 420 kDA exosome core complex, termed Eco-10, consists of the catalytically inactive Eco-9 complex, which is formed by the Nase PH-like ring and the S1 and KH ring. The Exo-9 complex associates with the Rrp44 (ribosomal RNA processing 44) exoribonuclease, which contains site of endonuclease (endo) and exonuclease (eco) activity. Note that the endonucleolytic site is exposed to solvent and not part of the internal channel.

Answer 101

Cytoplasmic Exo-10 complex binds the ATP-dependent SKI-complex which consist of a regulatory lid and an ATP-dependent helicase in the base. The nuclear Eco-10 complex also binds to ATP-independent regulators, including Rrp47 (ribosomal RNA processing 47), Rrp6 (an exonuclease!) and Mpp6 (M phase phosphoprotein 6)

Answer 102

- rRNA - tRNA - mRNA - sRNA (small RNAs) -> in E. coli 13 with known function (among them 4 housekeeping genes - tmRNA; 4.5 SRNA; RNase P RNA; 6S RNA) and probably 40-50 additional ones (e.g. as part of the CRISP/Cas system) -> trans-encoded sRNA -> antisense sRNA

Answer 103

- 16S, 23S and 5S rRNA and several tRNAs (depend on locus) are co-transcribed as one RNA molecule - the double-strand specific RNAse III claves after formation of partial double strands in this transcript, resulting in precursors of 16S, 23S, a tRNA and 5S + one or more tRNA - the 5S + tRNA precursors is than processed by RNAseE into individual molecules - 5' end maturation of 16S rRNA is performed by successive action of RNAse E and the related RNAse G; 3' end maturation is performed by an endonuclease - 5' end of 23S and 5S rRNA are maturated by unknown nuclease; 3' end are processed by exoribonuclease RNAse T - tRNA precursors coming from polycistronic RNA after RNAse E cleavage or individual precursors are processed at the 5' end by RNAse P - in E.coli, were all tRNA have their CCA in the gene, the 3' end is processed by RNAse T, RNAse PH or other RNAses; in B. subtitles, tRNA without CCA in their gene are trimmed by RNAse Z and CCA is added by tRNA nucleotide transferase

Answer 104

- structural RNA makes up 98 % of cellular RNA - quality control, of rRNA and tRNA uses similar RNAses as mRNA brake down systems - degraded under conditions of starvation by a pathway with steps similar to mRNA degradation - 50 % - up to 95 % of rRNA pool may become degraded, depending on organism and condition - regulating system unknown

Answer 105

- half-life of the bulk of E. coli mRNA is 2.4 min at 37 C (extremes 0.5-20 min) - 5' end has triphosphate cap - 3' ends of many bacterial mRNAs are sequestered in stem-loop structures that protect them from degradation; also protected are nascent transcripts - degradation is initiated by endonuclease (the 5' end dependent endonuclease RNAse E but sometimes also by RNAse III, G or P) - RNAse E cleaves preferentially single-stranded AU-rich regions with little secondary structure; RNAse III cleaves double-stranded regions - the cleaved products are digested by 3'->5' exonucleases (Nase II and polynucleotide phosphorylase (PNPase), probably also RNAse R) to products of 2-5 nt length. These are subject to degradation by oligoribonuclease - RNAse E, PNPase, the DEADbox RNA helicase B (RhIB) and other proteins from degradasome

Answer 106

Polyadenylation facilitates wegrationalisiere by 3' -> 5' exonuclesses, especially PNPase, as it provides a single-stranded starter region for the enzyme

Answer 107

degradation is initiated by endonuclease -> only high density for translating ribosomes on mRNA masks potential cleavage sites and thus protects against endonucleolytic attack -> partial decrease of ribosome density on the mRNA (e.g. due to poor Shine-Dalgarno sequences or proteins that block ribosome binding or PTC, or translational causing, or ....) may destabilize mRNA

Answer 108

- Endonucleolytic cleavage of mRNA leads to stalling of ribosomes due to lack of a termination codon. But also other circumstances (e.g. rare codons, low levels of antibiotic or other toxic conditions ....) may directly lead to ribosome stalling -> rescue system is needed - present in all eubacteria and in organelles of some eukaryotes - transfer-messenger RNA (tmRNA); encoded by ssrA gene in E. coli relieves stalled ribosomes with empty A site -> recycling of ribosomes and degradation of proteins resulting from this process - occurs after stalling in open reading frame or at termination codon

Answer 109

Alanyl-tmRNA recognizes ribosomes stalled at the end of a mRNA fragment and adds the alanine to the C terminus of the nascent polypeptide chain. Following mRNA swapping, the tmRNA ORF is translated, and RF1/RF2-mediated termination releases the tagged protein for degradation by cellular proteases and liberates the 30S and 50S subunits from the previously stalled ribosomes for new rounds of protein synthesis. Like normal tRNA the system needs alanyl-tRNA synthetase and EF-Tu. In addition SmpB is an essential and specific cofactor involved in the recognition of stalled ribosomes.

Answer 110

Active translating ribosomes are no tmRNA substrate because their A sites are usually occupied and because 3' mRNA and associated ribosomes or RNA polymerase (RNAP) would clash with tmRNA attempting to enter the A site. Ribosomes stalled at the 3' end of an mRNA have an unoccupied A site and may have an "open" mRNA entrance channel that allows the tmRNA ORF to be engaged.

Answer 111

- C-terminal tag in E.coli is AANDENYALAA - proteases involved in degradation: -> ClpXP (major cytoplasmic protease), ClpAP, FtsH, Tsp (periplasmic) have also roles -> degradation by ClpXP is enhanced by the activity of a ribosome-associated SspB, which binds specifically to tmRNA-tagged proteins -> SspB binds to the N-terinal AAND -> ClpXP recognizes the C-terminal ALAA

Answer 112

TA system (8 families in bacteria known): - a toxin and a labile antitoxin are expressed on a single operon; - down regulation of expression of the operon (arrest of transcription or translation or non-segregation of a plasmid containing the operon to a daughter cell) leads to arrest of cell growth Two families act on mRNA - Matze/PemK/Kid family of endoribonucleases attacks mRNA independent of ribosomes (present in bacteria) - ReIE cleaves mRNA engaged in translation with stalled ribosomes (due to starvation or lack of amino acids) (present in arches and bacteria)

Answer 113

AA sufficiency -> newly synthesized MazE and RelB bind to MazF and ReIE, resp., blocking their ribonuclease function. Complexes may also bind their promoter -> transcriptional autorepression. AA starvation -> block of MazE and ReIB synthesis + degradation of old proteins by Lon -> transcriptional derepression of the mazEF and reIBE operons; activation of MazF + ReIE ribonuclease functions. MazF cleaves mRNAs between ribosomes or free mRNAs. ReIE promotes cleavage of mRNAs at sites of ribosome stalling by binding to the ribosome A-site. Prolonged amino acid starvation may lead to a state of cell dormancy. Restoration of AA levels -> stalled ribosomes are rescued by tmRNA -> renewed synthesis of MazE and ReIB blocks MazF and ReIE activity once again -> resumption of cell growth adjusted to the new amino acid levels.

Answer 114

TRANSCRIPTION - 13 promoters - polycistronic transcripts (2 rRNAs, 25 tRNAs, 8 proteins) SPLICING - 3 genes with 13 introns; some code for maturates and endonucleases PROCESSING - cleavage and processing of polycistronic RNA - often longer 5' or 3' UTR MATURATION - no polyadenylation - CCA addition to tRNAs - rRNA with minimal base modification - no editing

Answer 115

TRANSCRIPTION - 3 promoters (H1, H2, L) - H-transcripts: 2 rRNAs, 14 tRNAs and 12 proteins - L: 8 tRNAs and one protein SPLICING - none PROCESSING - processing of polycistronic RNA - no cap (5' monophosphate) - nearly no 5' or 3' UTR MATURATION - H mRNAs + rRNAs polyadenylated - CCA addition to all tRNAs - base modification in rRNA and tRNAs - no editing

Answer 116

TRANSCRIPTION - 50-60 genes - many promoters - mono- and polycistronic RNAs - 3 rRNAs, 20 tRNAs, several proteins SPLICING - >20 introns - with one exception all group II type PROCESSING - processing occurs - 5' termini with three or diphosphate occur - long UTR MATURATION - transcripts not constitutively polyadenylated - base modification and CCA addition at all tRNA - most mRNAs contain editing sites (C to U)

Answer 117

- convergent (?) to bacterial degradosome - different systems in different organisms -> in yeast complex (mtEXO) made of helicase and a RNAse II- related exonuclease; involved in intron degradation, surveillance and indirectly (?) in processing of termini; specific dodecamer sequence at 3'-end binds protein factor and promotes stability -> human with helicase and PNP-like enzyme; no surveillance system (?) -> poly-A suggested to promote stability -> A. thaliana with helicase and PNP-like enzyme and RNAse II-related enzyme; Poly-A suggested as a signal for degradation

Answer 118

- probably dynamic structures, which provide a platform for the spatiotemporal regulation of the numerous processes required for mitochondrial gene expression, including RNA processing, maturation, ribosome assembly, and translation initiation - protein and RNA composition may change continuously depending on the progress of RNA processing and mitoribosome assembly

Answer 119

- spinach chloroplast degradasome has a 3' to 5' exonuclease: 100 RNP (quite similar to E. coli PNPase) an endonuclease: P67 (Nase E-like), and a 33 - RNP protein of unknown function

Answer 120

1) self-splicing group I introns 2) self-splicing group II introns 3) tRNA and/or Archean introns 4) spliceosoma. introns in nuclear pre-mRNA

Answer 121

- widely distributed in protist nuclear rRNA, fungal mitochondria, rare in bacteria and infrequently in viruses (including phages) and other organisms - 250 - 500 nt - self-splicing needs correct folding of the intron and the binding of an exogenous guanosine (exoG) cofactor to G-binding site of the catalytic core of the intron The cofactor attacks the 5' splice site (SS) and attaches to the intron resulting in the release of the upstream exon. exoG leaves the G-binding site and is replaced by the last nucleotide of the intron, which is always a G. Second step is initiated by an attack by the 3' end of the released exon on the 3' SS, which results in ligation of the exons and release of the intron.

Answer 122

Group I intron spread in DNA by homing endonuclease genes (HEGs) that invade non-critical regions (i.e. terminal loops) of group I introns and promote intron mobility by encoding highly site-specific homing endonucleases (Has). HEGs are themselves selfish genetic elements and the vast majority of group I introns in nature do not contain a HEG.

Answer 123

- genetic element that can excise themselves from pre-mRNA without the aid of proteins and that encode reverse transcriptase (RTs) and can insert themselves into new locations - present in mitochondria of plants and fungi, algae plastids, in about 25 % of eubacteria and in some archaebacteria - typical group II intron consist of a conserved intron RNA and an RT ORF

Answer 124

Domain V is probably catalytic core of the ribozyme ORF consists of: - RT domain - domain X (maturase) - domain D (DNA binding domain) - domain En (endonuclease) (D and En are missing in many introns)

Answer 125

- organellar introns behave more as splicing entity as most of them do not contain mobility ORF - bacterial group II introns appear to behave as retro elements more than introns; are associated with mobile DNAs (IS elements, plasmids, or pathogenicity islands), or sometimes inserted between genes and are rarely found in housekeeping genes - group II introns are believed to be ancestor of spliceosomal intron and non-Long Terminal Repeats in the eukaryotic nucleus

Answer 126

- a catalytic group II intron RNA functioning in the absence of an RT acquires the RT-maturase IEP leading to the ancestor of the three current intron lineages IIA, IIB and IIC - IIA and IIB introns present in the alphaproteobacterium ancestor of mitochondria would lead to massive invasion of the proeukaryotic host genome during eukaryogenesis, thus giving rise to spliceosomal introns

Answer 127

- found in the nuclear tRNA and in Archean tRNA, rRNA and mRNA - are enzymatically removed by a cut-and-rejoin mechanism that requires ATP and an endonuclease