Gene Expression http://www.ncbi.nlm.nih.gov/books/bv.fcgi?call=bv.View..ShowTOC&rid=mboc4.TOC&depth=2

 Prokaryotic

 Translation is concurrent with transcription

 No barrier restricts movement of transcript to translation apparatus

 Single RNA polymerase synthesizes all RNA species

 Eukaryotic

 Transcript must be processed

 Capping, splicing, polyA addition

 mRNA is sequestered as RNP in the nucleus, must be transported to cytoplasm

 Genes are often split - coding sequence is not contiguous

 3 different RNA polymerases required to synthesize RNA classes

Polycistronic Transcripts

Operon - gene cluster

DNA

mRNA Polycistronic transcript multiple genes

Examples: Proteins perform a Carbohydrate degradation coordinated function Amino acid biosynthesis

1 Eukaryotic Transcripts

 5’ 7-methylgaunosine cap structure

 Post-transcriptional modification - after ~ 25 nucleotides

 Prevents degradation by 5’ exonucleases

 Helps in the export from the nucleus

 Poly-adenylated tail

 Post-transcriptional modification

 Helps in stability of the mRNA

Mature transcript

Kinetoplastid Transcription

2

 Discovered by D. Baltimore - immunoglobin heavy chain

 Increases the diversity of protein repertoire

 Improper alternative splicing can lead to disease

Cis-Splicing Mechanism

3 Splicing is mediated by the

• Several steps in the splicing reaction require ATP

Splicesome mediated - simplified

 Composed of

 Small nuclear ribonucleoprotein

 Small nuclear U-rich RNA (snRNA)

 Each complexed with ~ 7 proteins

Highly simplified version

1. U1 base-pairs with the 5’ splice-site 2. U2 binds/pairs with the branch point; also pairs with U6 in the assembled spliceosome 3. U4 pairs with U6 in snRNPs, but releases during spliceosome assembly 4. U5 interacts with both (only 1-2 nt adjacent to ); helps bring exons together 5. U6 displaces U1 at the 5’ splice-site (pairs with nt in the intron); it also pairs with U2 in the catalytic center of the spliceosome

4 Trans-splicing: 1st discovered in trypanosomes

To date: ALL but 2 coding sequences are trans-spliced!

Gene A Gene B Gene C Gene D Gene E

DNA

Trans-splicing Polycistronic No evidence of transcript operons

SL RNA

AAAA AAAA Individual mRNAs each AAAA AAAA with a SL and poly A tail

AAAA

Comparison of cis- and trans-splicing

transesterification

Lariat Y-branch intermediate intermediate

transesterification

Intramolecular Intermolecular

5 Comparison of

New Technology - SMaRT

 Defects in alternative splicing can lead to human disease

 Use of artificial trans-splicing to “repair” and give rise to a functional mRNA

Spliceosome-mediated RNA Trans-splicing

www.intronn.com

Correcting at the pre-mRNA level!

6 kDNA - organized in a disk structure

kDNA

Kinetoplast

Nucleus

Mitochondrion

Kinetoplast is always associated with the flagellar basal body

kDNA components

Two types of catenated ring circles kDNA network

1. Maxicircle: ~23kb, 25 • Encode electron transport subunits. • Require extensive posttranscriptional editing

Maxicircle 3. Minicircle: 1kb, 5000 • Heterogeneous, 250 classes • Encode guide . 500nm 500nm kDNA is essential for the parasite survival

7 kDNA Replication Model

kDNA disk Primase Pol β -PAK DNA Ligase kα DNA Pol β DNA ligase kβ

Topo II SSE1

UMSBP

Pol IB Pol ID recruitment? Pol IC Pol IA ? kDNA repair?

What are the specialized roles?

Minicircle Replication Leading (L) strand

UMSBP

Unknown replicase + proteins

UMS Singly and Multiply Gapped Progeny UMS Lagging (H) strand

8 kDNA Replication Model

Trypanosomatid Mitochondrial RNA editing

 Single

 Unique mitochondrial DNA

 Catenated structure composed of mini- and maxicircles

 Size of molecules varies with species (15-80 kb) (1 - 2.5 kb)

 50 maxicircles/network

 5000-10,000 /network Maxicircle Minicircle 20 kb 1 kb  Minicircles were initially thought to be nonfunctional, just a structural component

9 Maxicircle sequence

 Initial sequencing of the T. brucei maxicircles demonstrated that it encoded apocytochrome b, subunits 1 and 2 of cytochrome c oxidase (cox) and some unassigned reading frames (MURFs) (some later turned out to be subunits of NADH dehydrogenase).  However some pseudogene features – e.g. cox2 had a –1 frameshift and this was conserved between kinetoplastid species.  Sequence determination of cox2 cDNA in 1984 showed an insertion at the precise position of the frameshift converting GA to UUGUAU.  This wasn’t accepted at first – there were 50 maxicircles and maybe one had the difference or the gene was encoded in the nucleus.  Extensive analysis showed no conventional cox2 genes existed in the nucleus or mitochondrion but a mechanism of adding in U’s was way too outlandish to be accepted at that time.

Maxicircle Sequence

 Sequencing of other mitochondrial cDNAs and their comparison to the genomic sequence showed not only the addition of U’s but also their deletion.  In 1986 the first CAUTIOUS paper on a “co- or post-transcriptional nucleotide insertion process” was published (Benne et al.,1986 Cell 46, 819-826 - 18 page paper).  Although the data showed deletion of one U, the authors didn’t dare to conclude that this form of editing could also occur.  Other groups of investigators found similar editing processes and the number of edited trypanosomatid RNAs expanded.  The mystery of missing AUG translational start codons was solved as these are provided by RNA editing by both addition and deletion of U’s

10 Mitochondrial RNA editing

 Cryptic mRNAs produced  mRNA sequence DOES NOT exactly Cell correspond with genomic DNA sequence * * ** ** *  Requires insertion of residues ** *** (u) or deletion (*) to create a * *** functional ORF **** *** * *  Extreme example is ND7 *** * ** *  >90% of mRNA is edited ***  Process is more active in *** ** ** ** procyclic form parasites **** **** *  Minicircles encode gRNAs (guide **** ** * *** RNAs) that act as templates for * * *** **** insertion and deletion (1991) * *** *  Process is essential (2001) * ** ******

 Demonstrated by gene silencing in Edited T. brucei ND7 mRNA bloodstream form parasites

Maxicircle Comparison

 Ribosomal RNA sequences ARE NOT edited

11 Insertional RNA editing

Primary transcript (Maxicircle encoded) 5' GCGGAGAAAAAAGAAAGGGUCUUUUAAUG (A)n ::|:|||| ||:|||||||| 3'-UUUUUUUUUU CAGAAAAUUACppp5' U A Guide RNA U A (Minicircle encoded) Poly(U) tail U C A A Anchor C U U U U A

Editing Edited mRNA

5' GCGGAGAAAAAAUGAAAUGUGUUGUCUUUUAAUG (A)n ::|:||||||||||||||:||||||||||||| 3'-UUUUUUUUUUUUUACUUUAUACAACAGAAAAUUACppp5'

Pan-editing of the L. tarentolae A6 mRNA

Precursor mRNA Edited mRNA

Precursor mRNA

Edited mRNA

Precursor mRNA

Edited mRNA

Precursor mRNA

Edited mRNA

12 Mechanism of RNA Editing

Insertion Deletion

RNA Editing Proteins

13 Mediated by Protein Complex

Metabolic T. brucei Life Cycle = adaptation

Bloodstream form non-dividing dividing fuel=? fuel=glucose mVSG coat VSG coat mito=? mito=“off”

Procyclic form dividing non-dividing fuel=amino acids fuel=glucose Procyclin coat VSG coat mito=“on” mito=“low”

14 Trypanosomatid Metabolism

 Cooperation among organelles for central metabolism

 Important Players

 Glycosomes Bloodstream form metabolism  Mitochondrion

 Cytoplasm

 Acidocalicosomes

Abundant microbodies = glycosome

Glycosomes

 What? Glycosomes are essential

 Microbody - single membrane for both BSF and Procyclics

 Divergent peroxisomes

 Peroxisomal metabolic diversity BSF - 90% of proteins content

 Contains most enzymes of is glycolytic glycolysis - unique

 Low permeability of membrane

Glycolysis  When?

 Not found in closest relative - Euglena sp.  Why?

 Metabolic flexibility

 How?

 Complicated - multiple mechanisms likely Purine salvage

15 Bloodstream Form Metabolism

 Most simplified form of metabolism in trypanosomes

 Glucose (sugars) are main source of energy

 Consumption and production of ATP is balanced INSIDE the glycosome

 Net ATP production occurs outside of the glycosome

 Major end-product - pyruvate

 Hexokinase (1), Phosphofructokinase (3) steps ARE NOT regulated

 Pyruvate kinase IS regulated (12)

 GPO/GAPDH shuttle - maintains redox balance

 Alternative Oxidase (CN- insensitive) Pyruvate excreted in host bloodstream *

Procyclic Form Metabolism

 Previously, thought there was complete TCA cycle function.

 Aconitase Knockout cell lines - aconitase is non-essential!!

 Glucose and amino acids (Pro, Thr) as energy source

 End products - Succinate, Acetate, Alanine

 Phosphoglycerate kinase is now cytosolic.

 Incomplete TCA cycle -

no complete oxidation to CO2  Branched electron transport - classical Cytochrome oxidase + alternative oxidase

16 Added complexity

 Anabolic functions as well

 Fatty acid synthesis

 Gluconeogenesis

 Branched electron transport

 Cytochrome oxidase

 Cyanide sensitive

 Alternative oxidase

 Cyanide insensitive gluco- neogenesis

Fatty Acid Synthesis - Primer

Dr. Kim Paul  Iterative elongation of acyl chains

 Growth of chain by 2 C

 Type I (Eukaryotic)

 Multiple enzymatic activities on a single large multifunctional protein

 Type II (Prokaryotic)

 Each activity is on a separate polypeptide

17 The Fatty Acid Dilemma

14  BSF cannot incorporate [ C]- acetate in FA.

 Parasite salvages FA, however free FA are not abundant in serum.

 Also enormous requirement for myristate (C14) for VSG GPI anchor structure.

 More classical Type II - synthesis of lipoic acid (α-keto DH complexes)

Third Mechanism - Elongation

18 Acidocalcisomes

 What?

 Membrane bound - acidic compartment

 Calcium storage

 Polyphosphate storage  When?

 Multiple lineages contain these organelles

 Trypanosomatids, Apicomplexans, fungi, algae, bacteria

 Mammals lack these organelles!

 Why?

 Potential role is for response to environmental stress

 Additional production of energy

Storage and Energy Generation?

19 RNAi in protist parasites

 Comparative biology reveals RNAi machinery in only a subset of protozoan parasites

20