Other RNA Processing Events

Chapter 16

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

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.

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

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

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

Processing Bacterial rRNA

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

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

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

Processing 3’-Ends of tRNA

RNase D, RNase BN, RNase T, RNase PH, RNase II and polynucleotide phosphorylase (PNPase)

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

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

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

Trans-Splicing

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

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

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

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

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

RNA Interference

• RNA interference occurs when a cell encounters dsRNA from:– Virus

– Transposon

– Transgene

• Trigger dsRNA is degraded into 21-23 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

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

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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