1. Other RNA Processing Events
Chapter 16

2. Ribosomal RNA Processing
rRNA genes of both eukaryotes and bacteria are transcribed as larger precursors must be processed to yield rRNAs of mature size
- Several different rRNA molecules are embedded in a long precursor and each must be cut out

3. Eukaryotic rRNA processing
The rRNA are separated by regions called nontranscribed spacers (NTSs).
Transcribed spacers - regions of the gene that are transcribed as part of rRNA precursor
- and then removed in processing of precursor to mature rRNA species.
NTSs are distinguished from transcribed spacers

4. Eukaryotic rRNA Processing
Ribosomal RNAs are made in eukaryotic nucleoli as precursors that must be processed to release mature rRNAs
Order of RNAs in the precursor is (fig: 16.2)
18S
5.8S
28S in all eukaryotes
Exact sizes of the mature rRNAs vary from one species to another
The rRNA are separated by regions called nontranscribed spacers (NTSs). NTSs are distinguished from transcribed spacers, regions of the gene that are transcribed as part of rRNA precursor and then removed in processing of precursor to mature rRNA species.

5. Eukaryotic rRNA Processing
rRNA processing – nucleolus – small nucleolar RNAs (snoRNAs) + proteins = Small nucleolar ribonucleoproteins (snoRNPs)
E1, E2 and E3 – interact with distinct regions in pre-rRNA

6. Bacterial rRNA Processing
Bacterial rRNA precursors contain tRNA and all 3 rRNA
rRNA are released from their precursors by RNase III and RNase E
RNase III is the enzyme that performs at least the initial cleavages that separate the individual large rRNAs
RNase E is another ribonuclease that is responsible for removing the 5S rRNA from the precursor

7. Processing Bacterial rRNA

8. Transfer RNA Processing
Transfer RNAs are made in all cells as overly long precursors
These must be processed by removing RNA at both ends
Nuclei of eukaryotes contain precursors of a single tRNA
In bacteria - precursor may contain one or more tRNA
- Sometimes a mixture of rRNAs and tRNAs

9. Cutting apart Polycistronic Precursors
In processing bacterial RNA that contain more than one tRNA
First step is to cut precursor up into fragments with just one tRNA each
Cutting between tRNAs in precursors having 2 or more tRNA
Cutting between tRNAs and rRNAs in precursors
Enzyme that performs both chores is the RNase III

10. Forming Mature 5’-Ends of tRNA
Extra nucleotides are removed from the 5’-ends of pre-tRNA in one step by an endonucleolytic cleavage catalyzed by RNase P
RNase P from bacteria and eukaryotic nuclei have a catalytic RNA subunit called M1 RNA
RNase P makes a cut at the site that becomes mature 5’-end of a tRNA

11. Processing 3’-Ends of tRNA
RNase D, RNase BN, RNase T, RNase PH, RNase II and polynucleotide phosphorylase (PNPase)

12. Forming mature 3’-Ends of tRNA
RNase II and polynucleotide phosphorylase cooperate
To remove most of extra nucleotides at the end of a tRNA precursor
RNases PH and T are most active in removing the last 2 nucleotides from RNA
RNase T is the major participant in removing very last nucleotide

13. Trans-Splicing
Splicing that occurs in all eukaryotic species is called cis-splicing because it involves 2 or more exons that exist together in the same gene
trans-splicing has exons that are not part of the same gene at all - may not even be on the same chromosome

14. The Mechanism of Trans-Splicing
Trans-splicing occurs in several organisms
Parasitic and free-living worms
First discovered in trypanosomes
Trypanosome mRNA are formed by trans-splicing between a short leader exon and any one of many independent coding exons

15. Trans-Splicing

16. Trans-Splicing Scheme
Branchpoint adenosine within the half-intron attached to the coding exon attacks the junction between the leader exon and its half-intron
Creates a Y-shaped intron-exon intermediate analogous to the lariat intermediate

17. Mechanism of Editing
Unedited transcripts can be found along with edited versions of the same mRNAs
Editing occurs in the poly(A) tails of mRNAs that are added posttranscriptionally
Partially edited transcripts have been isolated - always edited at their 3’-ends but not at their 5’-ends
Trypanosomatid mitochondria encode incomplete mRNA that must be edited before being translated
Editing occurs in the 3’5’ direction by successive action of one or more guide RNAs

18. Role of gRNA in Editing
Guide RNAs (gRNA) could direct the insertion and deletion of UMPs over a stretch of nucleotides in the mRNA
When editing is done, gRNA could hybridize near the 5’-end of newly edited region

19. Guide RNA Editing
5’-end of the first gRNA hybridizes to an unedited region at the 3’-border of editing I the pre-mRNA
The 5’-ends of the rest of the gRNAs hybridize to edited regions progressively closer to the 5’-end of the region to be edited in the pre-mRNA
All of these gRNAs provide A’s and G’s as templates for the incorporation of U’s missing from the mRNA

20. Mechanism of Removing U’s
Sometimes the gRNA is missing an A or G to pair with a U in the mRNA
In this case the U is removed
Mechanism of removing U’s involves
Cutting pre-mRNA just beyond U to be removed
Removal of U by exonuclease
Ligating the two pieces of pre-mRNA together
Mechanism of adding U’s uses same first and last step
Middle step involves addition of one or more U’s from UTP by TUTase

21. RNA Interference
RNA interference occurs when a cell encounters dsRNA from:
Virus
Transposon
Transgene
Trigger dsRNA is degraded into nt fragments (siRNAs) by an RNase III-like enzyme called Dicer
Double-stranded siRNA, with Dicer and Dicer-associated protein R2D2 form a complex called complex B

22. Complex B
Complex B delivers the siRNA to the RISC loading complex (RLC)
Separates 2 strands of siRNA
Transfers guide strand to RNA-induced silencing complex (RISC) that introduces a protein- Ago2

23. Complex B
The guide strand of siRNA base-pairs with target mRNA in the active site of PIWI domain of Ago2
Ago2 is an RNase H-like enzyme known as a slicer
Slicer cleaves the target mRNA in middle of the region of its base-pairing with the siRNA
ATP-dependent step has cleaved RNA ejected from RISC which then accepts a new molecule of mRNA for degradation

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